Combination of tim-4 antagonist and pd-1 antagonist and methods of use

ABSTRACT

Provided are methods and compositions for treating cancer using an effective amount of a PD-1 antagonist (e.g., an antibody) in combination with a TIM-4 antagonist (e.g., an antibody).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/US2017/040665, filed Jul. 5, 2017,which claims priority to U.S. Provisional Application No. 62/359,073,filed Jul. 6, 2016. The contents of the aforementioned applications arehereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 2, 2019, is named MXI_550USSequence_Listing.txt and is 45,365 bytes in size.

BACKGROUND

The National Cancer Institute has estimated that in the United Statesalone, 1 in 3 people will be struck with cancer during their lifetime.Moreover, approximately 50% to 60% of people contracting cancer willeventually succumb to the disease. Once tumor cells escape from theprimary site they passage through the lymphatic and/or circulatorysystem and ultimately a few establish at distant sites to givemetastases, and 95% of deaths from solid tumors in the developed worldare due to metastasis.

The widespread occurrence of cancer tumors underscores the need forimproved anticancer regimens. However, despite advances in multimodaltherapy, increases in overall survival in cancer patients have beenlimited. Accordingly, it is an object of the present invention toprovide improved methods for treating subjects with such tumors.

SUMMARY OF THE INVENTION

The present inventors have discovered for the first time thatco-administration of a TIM-4 antagonist (e.g., an antibody) and ananti-PD-1 antagonist (e.g., an antibody) effectively inhibits tumorgrowth in vivo, even synergistically. Accordingly, it is an object ofthe present invention to provide improved methods for treating subjectswith cancer. Specifically, it is an object of the invention to provideefficacious combination treatment regimens wherein a TIM-4 antagonist iscombined with an anti-PD-1 antagonist for the treatment of cancer.

In one aspect, the present invention provides a method for the treatmentof cancer in a subject by co-administering an effective amount of a PD-1antagonist and a TIM-4 antagonist.

Suitable TIM-4 antagonists for use in the methods of the invention,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies) and multivalent agents. The TIM-4antagonist can be a non-activating ligand or ligand binding partner(e.g., a small molecule, engineered PS emulator or soluble TIM-1, orother binding protein). In one embodiment, the TIM-4 antagonist is ananti-TIM4 antibody. In one embodiment, the TIM-4 antagonist is anantibody which binds to an epitope within the IgV domain of TIM-4. Inanother embodiment, the TIM-4 antagonist is an antibody such as 9F4,RMT4-53, RMT4-54, F31-563 or 21T112.

An exemplary anti-TIM-4 antibody contains heavy and light chainscomprising the heavy and light chain CDRs of 9F4, RMT4-53, RMT4-54,F31-563 or 21T112, and optionally comprises a framework region with atleast about 90% amino acid sequence identity with the framework regionof the corresponding antibody. Anti-TIM-4 antibodies may also compriseheavy and light chain variable domains that are at least about 90%, 95%or 99% identical with that of antibody 9F4, RMT4-53, RMT4-54, F31-563 or21T112. In certain embodiments, the anti-TIM-4 antagonist antibodycompetes for binding with, and/or binds to the same epitope on TIM-4 as,9F4, RMT4-53, RMT4-54, F31-563 or 21T112.

Suitable PD-1 antagonists for use in the methods of the invention,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.In another embodiment, the PD-1 antagonist is an antibody, such asMK-3475 or CT-011. In one embodiment, the PD-1 antagonist is a fusionprotein, e.g., an Fc fusion protein, such as AMP-244.

An exemplary anti-PD-1 antibody is 5C4 (referred to as 5C4 in WO2006/121168; also known as MDX-1106, ONO-4538, and nivolumab) comprisingheavy and light chains having the sequences shown in SEQ ID NOs: 11 and12, respectively, or antigen binding fragments and variants thereof. Inother embodiments, the antibody comprises the heavy and light chain CDRsor variable regions (VRs) of 5C4. Accordingly, in one embodiment, theantibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of5C4 having the sequence shown in SEQ ID NO: 13, and the CDR1, CDR2 andCDR3 domains of the V_(L) region of 5C4 having the sequence shown in SEQID NO:15. In another embodiment, the antibody comprises the heavy chainCDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ IDNOs: 17, 18, and 19, respectively, and the light chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NOs: 20, 21, and22, respectively. In another embodiment, the antibody comprises VHand/or VL regions having the amino acid sequences set forth in SEQ IDNO: 13 and/or SEQ ID NO: 15, respectively. In another embodiment, theantibody comprises the heavy chain variable (VH) and/or light chainvariable (VL) regions encoded by the nucleic acid sequences set forth inSEQ ID NO: 14 and/or SEQ ID NO: 16, respectively. In another embodiment,the antibody competes for binding with, and/or binds to the same epitopeon PD-1 as, the above-mentioned antibodies. In another embodiment, theantibody has at least about 90% variable region amino acid sequenceidentity with the above-mentioned antibodies (e.g., at least about 90%,95% or 99% variable region identity with SEQ ID NO: 13 or SEQ ID NO:15).

In one embodiment, the PD-1 antagonist is an anti-PD-L1 antibody, suchas MEDI4736 (also known as Anti-B7-H1) or MPDL3280A (also known asRG7446). An exemplary anti-PD-L1 antibody is 12A4 (referred to as 12A4in WO 2007/005874 and U.S. Pat. No. 7,943,743). In one embodiment, theantibody comprises the heavy and light chain CDRs or VRs of 12A4.Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2,and CDR3 domains of the VH region of 12A4 having the sequence shown inSEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains of the VL region of12A4 having the sequence shown in SEQ ID NO: 3. In another embodiment,the antibody comprises the heavy chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively,and the light chain CDR1, CDR2 and CDR3 domains having the sequences setforth in SEQ ID NOs: 8, 9, and 10, respectively. In another embodiment,the antibody comprises VH and/or VL regions having the amino acidsequences set forth in SEQ ID NO: 1 and/or SEQ ID NO: 3, respectively.In another embodiment, the antibody comprises the heavy chain variable(VH) and/or light chain variable (VL) regions encoded by the nucleicacid sequences set forth in SEQ ID NO: 2 and/or SEQ ID NO: 4,respectively. In another embodiment, the antibody competes for bindingwith, and/or binds to the same epitope on PD-L1 as, the above-mentionedantibodies. In another embodiment, the antibody has at least about 90%variable region amino acid sequence identity with the above-mentionedantibodies (e.g., at least about 90%, 95% or 99% variable regionidentity with SEQ ID NO: 1 or SEQ ID NO: 3).

In one embodiment, the invention provides a method of treating cancer ina subject, the method comprising administering to the subject aneffective amount of a PD-1 antagonist and a TIM-4 antagonist, wherein

-   -   (a) the PD-1 antagonist is an anti-PD-1 antibody comprising the        CDR1, CDR2 and CDR3 domains in a heavy chain variable region        having the sequence set forth in SEQ ID NO: 13, and the CDR1,        CDR2 and CDR3 domains in a light chain variable region having        the sequence set forth in SEQ ID NO: 15; and    -   (b) the TIM-4 antagonist is an antibody.

In another embodiment, the invention provides a method of treatingcancer in a subject, the method comprising administering to the subjectan effective amount of a PD-1 antagonist and a TIM-4 antagonist, wherein

-   -   (a) the PD-1 antagonist is an anti-PD-L1 antibody comprising the        CDR1, CDR2 and CDR3 domains in a heavy chain variable region        having the sequence set forth in SEQ ID NO: 1, and the CDR1,        CDR2 and CDR3 domains in a light chain variable region having        the sequence set forth in SEQ ID NO: 3; and    -   (b) the TIM-4 antagonist is an antibody.

The efficacy of the treatment methods provided herein can be assessedusing any suitable means. In one embodiment, the treatment produces atleast one therapeutic effect selected from the group consisting ofreduction in size of a tumor, reduction in number of metastasic lesionsover time, complete response, partial response, and stable disease. Inanother embodiment, administration of a PD-1 antagonist and a TIM-4antagonist results in at least a 1, 1.25, 1.50, 1.75, 2, 2.25, 2.50,2.75, 3, 3.25, 3.5, 3.75, or 4-fold reduction in tumor volume, e.g.,relative to treatment with the PD-1 antagonist or TIM-4 antagonistalone, or relative to tumor volume before initiation of treatment. Inanother embodiment, administration of the PD-1 antagonist and TIM-4antagonist results in at least a 1-fold, 2-fold, or more preferably a3-fold reduction in tumor volume, e.g., relative to treatment with thePD-1 antagonist or TIM-4 antagonist alone, or relative to tumor volumebefore initiation of treatment. In a further embodiment, administrationof a PD-1 antagonist and a TIM-4 antagonist results in tumor growthinhibition of at least 50%, 60%, 70% or 80%, e.g., relative to treatmentwith the PD-1 antagonist or TIM-4 antagonist alone, or relative to tumorvolume before initiation of treatment. In certain embodiments, tumorvolume is reduced by 50%, 60%, 70%, 80%, 90% or more, e.g., relative totumor size before initiation of the treatment.

The PD-1 antagonist and TIM-4 antagonist can be administered accordinglyto a suitable dosage, route (e.g., intravenous, intraperitoneal,intramuscular, intrathecal or subcutaneous). The antagonist and agonistcan also be administered according to any suitable schedule. Forexample, the antagonist and agonist can be simultaneously administeredin a single formulation. Alternatively, the antagonist and agonist canbe formulated for separate administration, wherein they are administeredconcurrently or sequentially. In one embodiment, the PD-1 antagonist isadministered prior to administration of the TIM-4 antagonist. In anotherembodiment, the TIM-4 antagonist is administered prior to administrationof the PD-1 antagonist. In a further embodiment, the TIM-4 antagonistand the PD-1 antagonist are administered simultaneously.

In one embodiment, the cancer is a cancer selected from the groupconsisting of carcinoma, sarcoma, blastoma, lymphoma and leukemia. Inone embodiment, the cancer is selected from the group consisting ofsmall-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, colorectal cancer, stomach cancer, colon carcinoma, andglioblastoma. In another embodiment, the cancer is selected from thegroup consisting of chronic myeloid leukemia, acute lymphoblasticleukemia, Philadelphia chromosome positive acute lymphoblastic leukemia(Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-smallcell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovariancancer, liver cancer, colorectal cancer, endometrial cancer, kidneycancer, prostate cancer, thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma multiforme, cervical cancer, stomach cancer,bladder cancer, hepatoma, breast cancer, colon carcinoma, and head andneck cancer, gastric cancer, germ cell tumor, pediatric sarcoma,sinonasal natural killer, multiple myeloma, acute myelogenous leukemia(AML), and chronic lymphocytic leukemia (CML).

Additional agents and therapies can be administered in combination withthe agonists and antagonists described herein. In one embodiment, themethods comprise administration of an additional therapeutic agent(e.g., a cyotoxin or chemotherapeutic agent.

Also provided herein are compositions comprising a PD-1 antagonist and aTIM-4 antagonist. In one embodiment, the antagonist is a ligand,antibody (e.g., monoclonal antibody or bispecific antibody) ormultivalent agent. In another embodiment, the PD-1 antagonist is ananti-PD-1 antibody comprising the heavy and light chain CDRs or VRs of5C4. In another embodiment, the PD-1 antagonist is an anti-PD-L1antibody comprising the heavy and light chain CDRs or VRs of 12A4.

Further provided are kits for treating a cancer in a subject, the kitcomprising:

-   -   (a) a dose of a PD-1 antagonist;    -   (b) a dose of a TIM-4 antagonist; and    -   (c) instructions for using the PD-1 antagonist and TIM-4        antagonist in the methods described herein. In one embodiment,        the TIM-4 antagonist is an antibody. In another embodiment, the        PD-1 antagonist is an antibody. In a particular embodiment, the        PD-1 antagonist is an anti-PD-1 antibody comprising the CDR1,        CDR2 and CDR3 domains in a heavy chain variable region having        the sequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and        CDR3 domains in a light chain variable region having the        sequence set forth in SEQ ID NO: 15. In another particular        embodiment, the PD-1 antagonist is an anti-PD-L1 antibody        comprising antibody comprises the CDR1, CDR2 and CDR3 domains in        a heavy chain variable region having the sequence set forth in        SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a light        chain variable region having the sequence set forth in SEQ ID        NO: 3.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of the amino acid sequences of human TIM-4isoforms 1 and 2.

FIG. 2 depicts the alignment of the amino acid sequences of cynomologousmonkey, mouse and human TIM-4 orthologs.

FIG. 3A-E are graphs depicting the myeloid cell populations in spleenand tumors from mice after administration of a control, a TIM-4antagonist antibody, a PD-1 antagonist antibody, or a combination of ananti-PD-1 antibody and an anti-TIM-4 antibody.

FIG. 4A-F are graphs depicting the CD8+ cell populations in spleen andtumors from mice after administration of a control, an TIM-4 antagonistantibody, an anti-PD-1 antibody, or a combination of an anti-PD-1antibody and an anti-TIM-4 antibody.

FIG. 5A-F are graphs depicting the CD4+ populations in spleen and tumorsfrom mice after administration of a control, an TIM-4 antagonistantibody, an anti-PD-1 antibody, or a combination of an anti-PD-1antibody and an anti-TIM-4 antibody.

FIG. 6A-D are graphs depicting tumor volume (mm³) in individual miceafter administration of a control, an TIM-4 antagonist antibody, ananti-PD-1 antibody, or a combination of an anti-PD-1 antibody and ananti-TIM-4 antibody.

FIG. 7A-B are graphs depicting the mean and median tumor volume (mm³) inmice after administration of a control, an TIM-4 antagonist antibody, ananti-PD-1 antibody, or a combination of an anti-PD-1 antibody and ananti-TIM-4 antibody.

FIG. 8 is a graph depicting the tumor volumes at day 28 in mice (mm³) inmice after administration of a control, an TIM-4 antagonist antibody, ananti-PD-1 antibody, or a combination of an anti-PD-1 antibody and ananti-TIM-4 antibody.

FIG. 9 (A) is a graph depicting the survival rates of mice afteradministration of a control, an TIM-4 antagonist antibody, an anti-PD-1antibody, or a combination of an anti-PD-1 antibody and an anti-TIM-4antibody in the CT26 animal model; and (B) is a table indicating themedian survival rate of each treatment group.

FIG. 10 is a graph depicting the percentage progression free survivalrates of mice after administration of a control, an TIM-4 antagonistantibody, an anti-PD-1 antibody, or a combination of an anti-PD-1antibody and an anti-TIM-4 antibody in the MC38 animal model.

FIG. 11 shows the mean tumor volume relative to the days post implant inthe mice that were in the experiment shown in FIG. 10.

FIG. 12A-C shows the individual tumor volumes relative to the days postimplant in the mice that were in the experiment shown in FIGS. 10 and11.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, the invention is based on the discovery thatco-administration of a TIM-4 antagonist (e.g., an antibody) and a PD-1antagonist (e.g., an antibody) effectively inhibits tumor growth invivo, even synergistically. Accordingly, the present invention providesa method for the treatment of cancer in a subject which comprisesadministering to a subject (e.g., human) an effective amount of a PD-1antagonist and a TIM-4 antagonist.

I. Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art, andconventional methods of immunology, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology are employed.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. The use of “or”or “and” means “and/or” unless stated otherwise. Furthermore, use of theterm “including” as well as other forms, such as “include”, “includes”,and “included”, is not limiting.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods. Unless otherwise indicated, all numbers expressing quantitiesof ingredients, properties such as molecular weight, reactionconditions, etc., used herein are to be understood as being modified bythe term “about”.

As used herein, the term “subject” or “patient” are used interchangeablyherein and refer to a mammal such as a human, mouse, rat, hamster,guinea pig, rabbit, cat, dog, monkey, cow, horse, pig and the like(e.g., a patient having cancer).

A “solid tumor” includes, for example, sarcoma, melanoma, carcinoma,prostate carcinoma, lung carcinoma, colon carcinoma, or other solidtumor cancer.

The terms “cancer”, “cancerous”, or “malignant” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. As used herein, the term includes pre-malignantas well as malignant cancers. Examples of cancer include, for example,leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particularexamples of such cancers include chronic myeloid leukemia, acutelymphoblastic leukemia, Philadelphia chromosome positive acutelymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-celllung cancer, non-small cell lung cancer, glioma, gastrointestinalcancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer, gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, multiple myeloma, acutemyelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

The term “carcinoma” as used herein refers to a type of cancer thatdevelops from epithelial cells. Specifically, a carcinoma is a cancerthat begins in a tissue that lines the inner or outer surfaces of thebody, and that generally arises from cells originating in the endodermalor ectodermal germ layer during embryogenesis. Various subtypes ofcarcinomas include, adenocarcinoma (featuring microscopicglandular-related tissue cytology, tissue architecture, and/orgland-related molecular products, e.g., mucin), squamous cell carcinoma(observable features and characteristics indicative of squamousdifferentiation, e.g., intercellular bridges, keratinization, squamouspearls), adenosquamous carcinoma (a mixed tumor containing bothadenocarcinoma and squamous cell carcinoma, wherein each of these celltypes comprise at least 10% of the tumor volume), anaplastic orundifferentiated carcinoma (a heterogeneous group of high-gradecarcinomas that feature cells lacking distinct histological orcytological evidence of any of the more specifically differentiatedneoplasms), large cell carcinoma (composed of large, monotonous roundedor overtly polygonal-shaped cells with abundant cytoplasm), small cellcarcinoma (cells are usually round and are less than approximately 3times the diameter of a resting lymphocyte and little evidentcytoplasm).

As used herein, the term “immune cell” refers to cells that play a rolein the immune response, including lymphocytes, such as B cells and Tcells; natural killer cells; myeloid cells, such as monocytes,macrophages, eosinophils, mast cells, basophils, and granulocytes.

The term “immune response” as used herein refers to a cellular immuneresponse, including T cell mediated and/or B cell mediated immuneresponses such as stimulation of T lymphocytes, macrophages, and/ornatural killer cells.

The terms “myeloid-derived suppressor cell” or “MDSC” are usedinterchangeably herein to refer to a heterogenous group of immune cellsfrom the myeloid lineage (a family of cells that originate from bonemarrow stem cells), to which dendritic cells, macrophages andneutrophils also belong. MDSCs strongly expand in pathologicalsituations such as chronic infections and cancer, as a result of analtered hematopoiesis. MDSCs are further classified into two subtypes,monocytic MDSCs and granulocytic MDSCs. MDSC suppressor function lies intheir ability to inhibit T cell proliferation and activation. Forexample, under chronic inflammatory conditions (viral and bacterialinfections) or cancer, myeloid differentiation is skewed towards theexpansion of MDSCs. These MDSCs infiltrate inflammation sites andtumors, where they inhibit immune responses by inhibiting T cells (e.g.,CD8+ T cells) and NK cells, for example. MDSCs also accelerateangiogenesis, tumor progression and metastasis through the expression ofcytokines and factors such as TGF-beta.

The terms “damage-associated molecular patterns” or “DAMPs” are usedinterchangeably herein to refer to intracellular molecules released byinjured tissues. DAMPs are molecules that have a physiological roleinside the cell, but acquire additional functions when exposed to theextracellular environment. For example, DAMPs alert the body aboutdanger, stimulate an inflammatory response, and finally promote theregeneration process. Beside their passive release by dead cells, someDAMPs can be secreted or exposed by living cells undergoing alife-threatening stress. DAMPs have been linked to inflammation andrelated disorders. DAMPs include, but are not limited to, histones,genomic DNA, HMGB1, IL1a, IL33, ATP, F-actin, cyclophilin A, HSPs, uricacid crystals, S100s, mitochondrial DNA, mitochondrial transcriptionfactor A, alreticulin.

The term “efferocytosis” refers to the process by which dying/dead cells(e.g., apoptotic or necrotic) are removed by phagocytic cells (e.g.,neurtrophils, monocytes, macrophages, mast cells and dendritic cells).During efferocytosis, phagocytes accumulate to the sites of apoptoticcells and the cell membrane of phagocytic cells engulfs the apoptoticcell, forming a large fluid-filled vesicle, the “effersome” containingthe dead cell. The effect of efferocytosis is that dead cells areremoved before their membrane integrity is breached and their contentsleak into the surrounding tissue. Efferocytosis triggers specificdownstream intracellular signal transduction pathways, for exampleresulting in anti-inflammatory, anti-protease and growth-promotingeffects. Conversely, impaired efferocytosis has been linked toautoimmune disease and tissue damage.

“Autophagy” or “autophagocytosis” is the natural, destructive mechanismthat disassembles, through a regulated process, unnecessary ordysfunctional cellular components. Autophagy allows the orderlydegradation and recycling of cellular components. During this process,targeted cytoplasmic constituents are isolated from the rest of the cellwithin a double-membraned vesicle known as an autophagosome. Theautophagosome then fuses with a lysosome and the contents are degradedand recycled. There are three different forms of autophagy that arecommonly described, namely macroautophagy, microautophagy andchaperone-mediated autophagy. In the context of disease, autophagy hasbeen seen as an adaptive response to stress which promotes survival,whereas in other cases it appears to promote cell death and morbidity.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent or combination of active agents to the subject with theobjective of reversing, alleviating, ameliorating, inhibiting, orslowing down or preventing the progression, development, severity orrecurrence of a symptom, complication, condition or biochemical indiciaassociated with a disease.

As used herein, “effective treatment” or “positive therapeutic response”refer to a treatment producing a beneficial effect, e.g., ameliorationof at least one symptom of a disease or disorder, e.g., cancer. Abeneficial effect can take the form of an improvement over baseline,i.e., an improvement over a measurement or observation made prior toinitiation of therapy according to the method. For example, a beneficialeffect can take the form of slowing, stabilizing, stopping or reversingthe progression of a cancer in a subject at any clinical stage, asevidenced by a decrease or elimination of a clinical or diagnosticsymptom of the disease, or of a marker of cancer. Effective treatmentmay, for example, decrease in tumor size, decrease the presence ofcirculating tumor cells, reduce or prevent metastases of a tumor, slowor arrest tumor growth and/or prevent or delay tumor recurrence orrelapse.

The term “effective amount” or “therapeutically effective amount” referto an amount of an agent or combination of agents that provides thedesired biological, therapeutic, and/or prophylactic result. That resultcan be reduction, amelioration, palliation, lessening, delaying, and/oralleviation of one or more of the signs, symptoms, or causes of adisease, or any other desired alteration of a biological system. Inreference to solid tumors, an effective amount comprises an amountsufficient to cause a tumor to shrink and/or to decrease the growth rateof the tumor (such as to suppress tumor growth) or to prevent or delayother unwanted cell proliferation. In some embodiments, an effectiveamount is an amount sufficient to delay tumor development. In someembodiments, an effective amount is an amount sufficient to prevent ordelay tumor recurrence. An effective amount can be administered in oneor more administrations. The effective amount of the drug or compositionmay: (i) reduce the number of cancer cells; (ii) reduce tumor size;(iii) inhibit, retard, slow to some extent and may stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and may stop tumor metastasis; (v) inhibit tumor growth; (vi)prevent or delay occurrence and/or recurrence of tumor; and/or (vii)relieve to some extent one or more of the symptoms associated with thecancer. In one example, an “effective amount” is the amount of a PD-1antagonist (e.g., an antibody) and TIM-4 antagonist antibody (e.g., anantibody), in combination, to effect a significant decrease in cancer orslowing of progression of cancer, such as an advanced solid tumor. Aneffective amount of the combination therapy is administered according tothe methods described herein in an “effective regimen” which refers to acombination of the PD-1 antagonist and the TIM-4 antagonist, wherein theorder of administration and dosage frequency is adequate to effecttreatment.

“Optimal biologic dose (OBD)” is defined as the minimum dose of an agentor combination of agents that gives the most optimal and lasting in vivoresponse without clinically unacceptable toxicity. Toxicity andtherapeutic efficacy can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD50/ED50.

As used herein, the terms “synergy”, “therapeutic synergy”, and“synergistic effect” refer to a phenomenon where treatment of patientswith a combination of therapeutic agents (e.g., PD-1 antagonist incombination with TIM-4 antagonist) manifests a therapeutically superioroutcome to the outcome achieved by each individual constituent of thecombination used when used alone (see, e.g., T. H. Corbett et al., 1982,Cancer Treatment Reports, 66, 1187). In this context a therapeuticallysuperior outcome includes one or more of the following (a) an increasein therapeutic response that is greater than the sum of the separateeffects of each agent alone at the same dose as in the combination; (b)a decrease in the dose of one or more agents in the combination withouta decrease in therapeutic efficacy; (c) a decrease in the incidence ofadverse events while receiving a therapeutic benefit that is equal to orgreater than the monotherapy of each agent at the same dose as in thecombination, (d) a reduction in dose-limiting toxicities while receivinga therapeutic benefit that is greater than the monotherapy of eachagent; (e) a delay or minimization of the induction of drug resistance.In xenograft models, a combination, used at its maximum tolerated dose,in which each of the constituents will be present at a dose generallynot exceeding its individual maximum tolerated dose, manifeststherapeutic synergy when decrease in tumor growth achieved byadministration of the combination is greater than the value of thedecrease in tumor growth of the best constituent when the constituent isadministered alone. Synergism of a drug combination may be determined,for example, according to the combination index (CI) theorem ofChou-Talalay (Chou et al., Adv. Enzyme Regul. 1984; 22:27-55; Chou,Cancer Res. 2010; 70(2):440-446).

“Free of cancer” or “disease free” or NED (No Evidence of Disease) meansthat the patient has demonstrated a clinical response induced bytreatment with the current standard of care therapies. By “clinicalresponse,” it is meant that the clinical signs, radiological signs, andsymptoms of cancer have been significantly diminished or havedisappeared entirely based on clinical diagnostics, although cancerouscells can still exist in the body. Thus, it is contemplated thatclinical response encompasses partial and complete response. Thepresence of residual cancer cells can be enumerated by assays such asCTC (Circulating Tumor Cells) and can be predictive of recurrence.

“Relapse” or “recurrence” or “resurgence” are used interchangeablyherein, and refer to the radiographic diagnosis of return, or signs andsymptoms of return of cancer after a period of improvement or response.

As used herein, the term “antagonist” refers to a molecule which blocks(e.g., reduces or prevents) a biological activity. The term “inhibit” or“inhibition” means to reduce by a measurable amount.

As used herein, the term “ligand” refers to a molecule that forms acomplex with a biomolecule (e.g., a receptor) to serve a biologicalpurpose. In a narrower sense, is a signal triggering molecule, bindingto a site on a target protein. The binding occurs by intermolecularforces, such as ionic bonds, hydrogen bonds and van der Waals forces.The docking (association) is usually reversible (dissociation). Actualirreversible covalent binding between a ligand and its target moleculeis rare in biological systems. Ligand binding to a receptor (receptorprotein) alters its chemical conformation (three dimensional shape). Theconformational state of a receptor protein determines its functionalstate.

The “level” of a protein refers to the amount of protein in a sample asdetermined using any method known in the art for measuring proteinlevels, including electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitationreactions, absorption spectroscopy, colorimetric assays,spectrophotmetric assays, flow cytometry, immmunodiffusion, solutionphase assay, immunoelectrophoresis, Western blotting, radioimmunoassay(MA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescentassays and electrochemiluminescence immunoassays.

The term “sample” refers to a collection of fluids, cells or tissuesisolated from a subject. Biological fluids are typically liquids atphysiological temperatures and may include naturally occurring fluidspresent in, withdrawn from, expressed or otherwise extracted from asubject or biological source. Examples of biological fluids includeblood, serum, serosal fluids, plasma, lymph, urine, cerebrospinal fluid,saliva, ocular fluids, cystic fluid, tear drops, feces, sputum, mucosalsecretions, vaginal secretions, gynecological fluids, ascites fluidssuch as those associated with non-solid tumors, fluids of the pleural,pericardial, peritoneal, abdominal and other body cavities, fluidscollected by bronchial lavage and the like.

The term “control sample”, as used herein, refers to any clinicallyrelevant control sample, including, for example, a sample from a healthysubject or a sample made at an earlier time point from the subject to beassessed. For example, the control sample can be a sample taken from thesubject prior to onset of cancer, at an earlier stage of disease, orbefore the administration of treatment or of a portion of treatment.

As used herein, the term “antibody” includes whole antibodies and anyantigen binding fragment (i.e., “antigen-binding fragments” (also knownas “antigen-binding portions”)). Whole antibodies are glycoproteinscomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, C_(H)L C_(H)2 and C_(H)3. Each light chain is comprisedof a light chain variable region (abbreviated herein as V_(L)) and alight chain constant region. The light chain constant region iscomprised of one domain, C_(L). The V_(H) and V_(L) regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each V_(H) andV_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. The term “antibody” also encompasseschimeric antibodies, humanized antibodies, fully human antibodies, aswell as multimeric forms of antibodies, such as minibodies, bis-scFv,diabodies, triabodies, tetrabodies and chemically conjugated Fab′multimers.

The term “antibody fragment” (also referred to as “antigen-bindingfragment” or “antigen-binding portion”), as used herein, refers to oneor more fragments of an antibody that retain the ability to specificallybind to an antigen. It has been shown that the antigen-binding functionof an antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) a F(ab′)2 fragment, a bivalent fragment is essentially aFab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Pauled., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the V_(H) andC_(H)1 domains; (v) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (vi) a dAb fragment (Ward etal., (1989) Nature 341:544-546), which consists of a V_(H) domain; (vii)an isolated complementarity determining region (CDR); and (viii) ananobody (also known as a single-domain antibody (sdAb)), which is aheavy chain variable region containing a single variable domain and twoconstant domains. Single domain antibodies include V_(H)H fragments(single-domain antibodies engineered from heavy-chain antibodies foundin camelids, as well as VNAR fragments (single-domain antibodiesobtained from heavy-chain antibodies (IgNAR, ‘immunoglobulin new antigenreceptor’) of cartilaginous fishes).

“Antigen binding scaffolds” are proteins that bind specifically to atarget (or antigen) or epitope, such as proteins comprising an Ig foldor an Ig-like fold. Antibodies or antigen binding fragments thereof arealso antigen binding scaffolds. Antigen binding scaffolds can bemonovalent, multivalent, e.g., bivalent, trivalent, tetravalent, or bind5, 6 or more epitopes. Multivalent antigen binding scaffolds can bemonospecific or multispecific, i.e., binding to multiple (at least 2, 3,4 or 5) epitopes that are different from one another. For example, amultivalent monospecific antigen binding scaffold is a protein thatbinds to at least 2, 3, 4 or 5 identical epitopes, and may be a proteincomprising at least 2, 3, 4 or 5 identical antigen binding portions. Forexample, TIM-4 binding scaffolds may comprise 2-10, e.g., 2-6, 2-5, 2-4or 2-3 TIM-4 binding portions, which may be the same or different fromone another.

A multivalent antibody includes antibodies comprising at least 2, 3, 4,5, 6, 7, 8, 9, 10 or more antigen binding portions of antibodies, whichantigen binding portions may comprise a portion of a heavy chain and aportion of a light chain. An antigen binding portion may be on a singlepolypeptide or comprise more than one polypeptide. For example, amultivalent antibody may comprise from 2-10 antigen binding portions,which may be the same or different from each other. A multivalentantibody may be monospecific or multispecific. A multispecific antibodymay be bispecific, trispecific, tetraspecific or bind to 5 or moredifferent epitopes.

Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingfragment” of an antibody. These antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

As used herein, an antigen binding scaffold that “specifically binds” toan antigen or epitope thereof is an antigen binding scaffold that bindsto the antigen or epitope thereof with a K_(D) of 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹²M or less. For example, an antigen binding scaffold thatspecifically binds to TIM-4 is an antigen binding scaffold that binds toTIM-4 with a K_(D) of 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M or less. For example,an antibody that “specifically binds to human PD-1” or “specificallybinds to human PD-L1” is intended to refer to an antibody that binds tohuman PD-1 or PD-L1, respectively, with a K_(D) of 10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M or less. An antigen binding scaffold that comprises 2 or moreregions binding to an antigen or epitope may bind specifically to theantigen or epitope even it has a lower affinity of binding to theantigen or epitope than the ranges provided above, as it will bind tothe antigen or epitope with increased avidity.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody” as used herein, refers to refers to anantibody from a population of substantially homogeneous antibodies thatdisplay a single binding specificity and affinity for a particularepitope, except for possible variants that may arise during productionof the monoclonal antibody, such variants generally being present inminor amounts. Accordingly, the term “human monoclonal antibody” or“monoclonal antibody composition” refers to an antibody which hasvariable and optional constant regions derived from human germlineimmunoglobulin sequences. The modifier “monoclonal” indicates thecharacter of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies described herein may be made by avariety of techniques. In one embodiment, human monoclonal antibodiesare produced by a hybridoma which includes a B cell obtained from atransgenic non-human animal, e.g., a transgenic mouse, having a genomecomprising a human heavy chain transgene and a light chain transgenefused to an immortalized cell.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods of determiningspatial conformation of epitopes include techniques in the art and thosedescribed herein, for example, x-ray crystallography, HDX-MS and2-dimensional nuclear magnetic resonance (see, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope,” with reference to two or moreantibodies, means that the antibodies compete for binding to an antigenand bind to the same, overlapping, or encompassing continuous ordiscontinuous segments of amino acids. Those of skill in the artunderstand that the phrase “binds to the same epitope” does notnecessarily mean that the antibodies bind to exactly the same aminoacids. The precise amino acids to which the antibodies bind can differ.For example, a first antibody can bind to a segment of amino acids thatis completely encompassed by the segment of amino acids bound by asecond antibody. In another example, a first antibody binds one or moresegments of amino acids that significantly overlap the one or moresegments bound by the second antibody. For the purposes herein, suchantibodies are considered to “bind to the same epitope.” Two antibodies“bind to the same epitope” as determined by a given method, e.g.,HDX-MS, crystallography or target mutational analysis, requires that thesame amino acids are identified for both antibodies by one or more givenmethods.

Accordingly, also, encompassed by the present invention are antibodiesthat bind to an epitope which comprises all or a portion of an epitoperecognized by the particular antibodies described herein (e.g., the sameor an overlapping region or a region between or spanning the region) orthat bind to the same epitope as determined by a given method, e.g.,HDX-MS, crystallography or target mutational analysis.

Also encompassed by the present invention are antibodies that competefor binding with the antibodies described herein, and optionally bindthe same epitope. Antibodies that compete for binding can be identifiedusing routine techniques. Such techniques include, for example, animmunoassay, which shows the ability of one antibody to block thebinding of another antibody to a target antigen, i.e., a competitivebinding assay. Competitive binding is determined in an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: Biacore, flow cytometry, solid phasedirect or indirect radioimmunoassay (MA), solid phase direct or indirectenzyme immunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using 1-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

Epitope mapping methods also include x-ray analyses of crystals ofantigen:antibody complexes which provides atomic resolution of theepitope. Other methods monitor the binding of the antibody to antigenfragments or mutated variations of the antigen where loss of binding dueto a modification of an amino acid residue within the antigen sequenceis often considered an indication of an epitope component. In addition,computational combinatorial methods for epitope mapping can also beused. These methods rely on the ability of the antibody of interest toaffinity isolate specific short peptides from combinatorial phagedisplay or yeast display peptide libraries. The peptides are thenregarded as leads for the definition of the epitope corresponding to theantibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

Chimeric molecules (or fusion molecules) comprising an antigen bindingdomain, or equivalent, fused to another polypeptide or molecule are alsoencompassed by the present invention. For example, the polypeptides maybe fused or conjugated to an antibody Fc region, or portion thereof(e.g., an Fc fusion protein). The antibody portion fused to apolypeptide may comprise the constant region, hinge region, C_(H)1domain, C_(H)2 domain, and C_(H)3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, and 5,112,946; EP 307,434; EP 367,166; PCTPublication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., Proc.Natl. Acad. Sci. USA, 88:10535-10539 (1991); Zheng et al., J. Immunol.,154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341 (1992).

As used herein, the term “immunoconjugate” refers to an antibody linkedto a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope.When conjugated to a cytotoxin, these antibody conjugates are referredto as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agentthat is detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Antibodies use in the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating cancer.

Immunoconjugates can be used to modify a given biological response, andthe drug moiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, an enzymatically active toxin, or active fragmentthereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin; a protein such as tumor necrosis factor or interferon-γ; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

As used herein, the term “multivalent” refers to a recombinant moleculethat incorporates more than two biologically active segments. Theprotein fragments forming the multivalent molecule optionally may belinked through a polypeptide linker which attaches the constituent partsand permits each to function independently.

As used herein, “functional equivalents” refer to peptides orpolypeptides (e.g., antibodies or antigen binding portion thereof) whichmaintain a substantial amount of their original immunological activity.For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. It isthus contemplated that various changes may be made in the amino acidsequences of the disclosed compositions without appreciable loss oftheir biological utility or activity. Amino acid substitutions may bebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art, and such immunologically functional equivalents arealso encompassed within the present invention.

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by a sequence alignment program, such asBLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR), in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

II. PD-1 Antagonists

As used herein, the terms “Protein PD-1,” “PD-1,” PD1,” “PDCD1” are usedinterchangeably with “Programmed Death 1,” “Programmed Cell Death 1.”The complete human PD-1 sequence can be found under GenBank AccessionNo. U64863 (SEQ ID NO:23).

As used herein, the terms “PD-L1”, “PDL1”, “PDCD1L1”, “PDCD1LG1”,“CD274”, “B7 homolog 1”, “B7-H1”, “B7-H”, and “B7H1” are usedinterchangeably with “Programmed Cell Death 1 Ligand 1.” The completehuman PD-L1 amino acid sequence—isoform a precursor—can be found underGenBank Accession No. NP_054862.1 (SEQ ID NO:24). The complete humanPD-L1 amino acid sequence—isoform b precursor—can be found under GenBankAccession No. NP_001254635.1 (SEQ ID NO:25).

The protein Programmed Death 1 (PD-1) is an inhibitory member of theCD28 family of receptors, that also includes CD28, CTLA-4, ICOS andBTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells(Agata et al., supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14:391779-82; Bennett et al. (2003) J Immunol 170:711-8). The initialmembers of the family, CD28 and ICOS, were discovered by functionaleffects on augmenting T cell proliferation following the addition ofmonoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansenet al. (1980) Immunogenics 10:247-260). PD-1 was discovered throughscreening for differential expression in apototic cells (Ishida et al.(1992) EMBO J 11:3887-95). The other members of the family, CTLA-4 andBTLA, were discovered through screening for differential expression incytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS andCTLA-4 all have an unpaired cysteine residue allowing forhomodimerization. In contrast, PD-1 is suggested to exist as a monomer,lacking the unpaired cysteine residue characteristic in other CD28family members.

The PD-1 protein is a 55 kDa type I transmembrane protein that is partof the Ig gene superfamily (Agata et al. (1996) Int Immunol 8:765-72).PD-1 contains a membrane proximal immunoreceptor tyrosine inhibitorymotif (ITIM) and a membrane distal tyrosine-based switch motif (ITSM)(Thomas, M. L. (1995) J Exp Med 181:1953-6; Vivier, E and Daeron, M(1997) Immunol Today 18:286-91). Although structurally similar toCTLA-4, PD-1 lacks the MYPPPY motif (SEQ ID NO: 27) that is critical forB7-1 and B7-2 binding.

Consistent with PD-1 being an inhibitory member of the CD28 family, PD-1deficient animals develop various autoimmune phenotypes, includingautoimmune cardiomyopathy and a lupus-like syndrome with arthritis andnephritis (Nishimura et al. (1999) Immunity 11:141-51; Nishimura et al.(2001) Science 291:319-22). Additionally, PD-1 has been found to play arole in autoimmune encephalomyelitis, systemic lupus erythematosus,graft-versus-host disease (GVHD), type I diabetes, and rheumatoidarthritis (Salama et al. (2003) J Exp Med 198:71-78; Prokunina andAlarcon-Riquelme (2004) Hum Mol Genet 13:R143; Nielsen et al. (2004)Lupus 13:510). In a murine B cell tumor line, the ITSM of PD-1 was shownto be essential to block BCR-mediated Ca²⁺-flux and tyrosinephosphorylation of downstream effector molecules (Okazaki et al. (2001)PNAS 98:13866-71).

Two ligands for PD-1 have been identified, PD-L1 and PD-L2, that havebeen shown to downregulate T cell activation upon binding to PD-1(Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) NatImmunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43). BothPD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind toother CD28 family members. PD-L1 is abundant in a variety of humancancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction betweenPD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes,a decrease in T-cell receptor mediated proliferation, and immune evasionby the cancerous cells (Dong et al. (2003) 1 Mol. Med. 81:281-7; Blanket al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al.(2004) Clin. Cancer Res. 10:5094-100). Immune suppression can bereversed by inhibiting the local interaction of PD-1 with PD-L1, and theeffect is additive when the interaction of PD-1 with PD-L2 is blocked aswell (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brownet al. (2003) 1 Immunol. 170:1257-66).

The methods of the present invention involve the use of a PD-1antagonist (e.g., an antibody) in combination with a TIM-4 antagonist(e.g., an antibody), for treating cancer. Accordingly, PD-1 antagonistsof the invention bind to ligands of PD-1 and interfere with, reduce, orinhibit the binding of one or more ligands to the PD-1 receptor, or binddirectly to the PD-1 receptor, without engaging in signal transductionthrough the PD-1 receptor. In one embodiment, the PD-1 antagonist bindsdirectly to PD-1 and blocks PD-1 inhibitory signal transduction. Inanother embodiment the PD-1 antagonist binds to one or more ligands ofPD-1 (e.g., PD-L1 and PD-L2) and reduces or inhibits the ligand(s) fromtriggering inhibitory signal transduction through the PD-1. In oneembodiment, the PD-1 antagonist binds directly to PD-L1, inhibiting orpreventing PD-L1 from binding to PD-1, thereby blocking PD-1 inhibitorysignal transduction.

PD-1 antagonists used in the methods and compositions of the presentinvention include PD-1 binding scaffold proteins and include, but arenot limited to, PD-1 ligands, antibodies and multivalent agents. In aparticular embodiment, the antagonist is a fusion protein, such asAMP-224. In another embodiment, the antagonist is an anti-PD-1 antibody(“PD-1 antibody”). Anti-human-PD-1 antibodies (or VH and/or V_(L)domains derived therefrom) suitable for use in the invention can begenerated using methods well known in the art. Alternatively, artrecognized anti-PD-1 antibodies can be used. For example, antibodiesnivolumab (OPDIVO™), MK-3475 (pembrolizumab (KEYTRUDA™), PDR001 orCT-011 can be used. Additionally, monoclonal antibodies 5C4, 17D8, 2D3,4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the teachings ofwhich are hereby incorporated by reference, can be used. Antibodies thatcompete with any of these art-recognized antibodies for binding to PD-1also can be used.

An exemplary anti-PD-1 antibody is 5C4 comprising heavy and light chainshaving the sequences shown in SEQ ID NOs: 11 and 12, respectively, orantigen binding fragments and variants thereof. In other embodiments,the antibody comprises the heavy and light chain CDRs or variableregions of 5C4. Accordingly, in one embodiment, the antibody comprisesthe CDR1, CDR2, and CDR3 domains of the VH of 5C4 having the sequenceset forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains of theVL of 5C4 having the sequences set forth in SEQ ID NO: 15. In anotherembodiment, the antibody comprises CDR1, CDR2 and CDR3 domains havingthe sequences set forth in SEQ ID NOs: 17, 18, and 19, respectively, andCDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ IDNOs: 20, 21, and 22, respectively. In another embodiment, the antibodycomprises VH and/or VL regions having the amino acid sequences set forthin SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively. In anotherembodiment, the antibody comprises the heavy chain variable (VH) and/orlight chain variable (VL) regions encoded by the nucleic acid sequencesset forth in SEQ ID NO: 14 and/or SEQ ID NO: 16, respectively. Inanother embodiment, the antibody competes for binding with and/or bindsto the same epitope on PD-1 as the above-mentioned antibodies. Inanother embodiment, the antibody has at least about 90% variable regionamino acid sequence identity with the above-mentioned antibodies (e.g.,at least about 90%, 95% or 99% variable region identity with SEQ ID NO:13 or SEQ ID NO: 15).

In certain embodiments, the PD1 antibodies exhibit one or more desirablefunctional properties, such as high affinity binding to PD-1, e.g.,binding to human PD-1 with a K_(D) of 10⁻⁷ M or less; lack ofsignificant cross-reactivity to other CD28 family members, e.g., CD28,CTLA-4 and ICOS; the ability to stimulate T cell proliferation in amixed lymphocyte reaction (MLR) assay; the ability to increase IFN-γand/or IL-2 secretion in an MLR; the ability to inhibit binding of oneor more PD-1 ligands (e.g., PD-L1 and/or PD-L2) to PD-1; the ability tostimulate antigen-specific memory responses; the ability to stimulateantibody responses and/or the ability to inhibit growth of tumor cellsin vivo.

In another embodiment, the PD-1 antagonist is an anti-PD-L1 antibody.Anti-human-PD-L1 antibodies (or VH and/or VL domains derived therefrom)suitable for use in the invention can be generated using methods wellknown in the art. Alternatively, art recognized anti-PD-L1 antibodiescan be used. For example, MEDI4736 (also known as Anti-B7-H1; durvalumab(IMFINZI™)), MPDL3280A (atezolizumab (TECENTRIQ™) also known as RG7446),and avelumab (BAVENCIO™) and can be used. Additionally, monoclonalantibodies 12A4, 3G10, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4described in WO 2007/005874 and U.S. Pat. No. 7,943,743, the teachingsof which are hereby incorporated by reference, can be used. Antibodiesthat compete with any of these art-recognized antibodies for binding toPD-L1 also can be used.

An exemplary anti-PD-L1 antibody is 12A4 (WO 2007/005874 and U.S. Pat.No. 7,943,743). In one embodiment, the antibody comprises the heavy andlight chain CDRs or VRs of 12A4. Accordingly, in one embodiment, theantibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of12A4 having the sequence shown in SEQ ID NO: 1 and the CDR1, CDR2 andCDR3 domains of the VL region of 12A4 having the sequence shown in SEQID NO: 3. In another embodiment, the antibody comprises the heavy chainCDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ IDNOs: 5, 6, and 7, respectively, and the light chain CDR1, CDR2 and CDR3domains having the sequences set forth in SEQ ID NOs: 8, 9, and 10,respectively. In another embodiment, the antibody comprises VH and/or VLregions having the amino acid sequences set forth in SEQ ID NO: 1 and/orSEQ ID NO: 3, respectively. In another embodiment, the antibodycomprises the heavy chain variable (VH) and/or light chain variable (VL)regions encoded by the nucleic acid sequences set forth in SEQ ID NO: 2and/or SEQ ID NO: 4, respectively. In another embodiment, the antibodycompetes for binding with, and/or binds to the same epitope on PD-L1 as,the above-mentioned antibodies. In another embodiment, the antibody hasat least about 90% variable region amino acid sequence identity with theabove-mentioned antibodies (e.g., at least about 90%, 95% or 99%variable region identity with SEQ ID NO: 1 or SEQ ID NO: 3).

Anti-PD-1 or anti-PD-L1 antibodies may bind to PD-1 or PD-L1,respectively, with a K_(D) of 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰ M, 10⁻¹⁰ M or less.

In certain embodiments, an anti-PD-1 or anti-PD-L1 antibody is an IgGantibody, such as an IgG1, IgG2 or IgG4 antibody. In specificembodiments, an anti-PD-1 or PD-11 antibody has an effectorless constantregion. Anti-PD-1 or PD-L1 antibodies may be IgG4 antibodies, e.g., IgG4antibodies having an S228P mutation.

III. TIM-4 Antagonists

As used herein, the terms “TIM-4” and “TIMD-4”, also known as “T-cellimmunoglobulin and mucin domain-containing protein 4”, “T-cellimmunoglobulin mucin receptor 4,” “T-cell membrane protein 4” and“SMUCKLER” are used interchangeably. The complete amino acid andnucleotide sequences of isoform-1 of human TIM-4 can be found underGenBank Accession Nos. NP_612388.2 and NM_138379.2, respectively (SEQ IDNOs: 26-27). The complete amino acid and nucleotide sequences ofisoform-2 of human TIM-4 can be found under GenBank Accession Nos.NP_001140198.1 and NM_001146726.1, respectively (SEQ ID NOs: 28-29)(Jones et al., Int Arch Allergy Immunol. 2006; 141(4):331-6). Analignment of the two human isoforms is depicted in FIG. 1.

The amino acid sequences of TIM-4 isoforms from cynomologus monkey canbe found under GenBank Accession Nos. XP_005558436 and EHH54702, and theamino acid sequences for murine TIM-4 isoforms can be found underGenBank Accession Nos. NP_848874 and NP_599009. An alignment of theamino acid sequences of mouse, cynomolgus monkey and human TIM-4polypeptides is provided in FIG. 2, and the percent identity of thefull-length and IgV regions of human, cynomolgus and murine orthologsare set forth in Table 1.

TABLE 1 TIM-4 Orthologs hTIM4-FL hTIM4 IgV Cyno 87% 93% Mouse 49% 64%

The protein “TIM-4” is type I membrane protein that is a member ofT-cell immunoglobulin and mucin domain-containing (TIM) family. Thehuman TIM family contains three members (TIM-1, TIM-3 and TIM-4) onhuman chromosome 5q33.2, located in a chromosomal region that has beenlinked with asthma, allergy and autoimmunity. TIM proteins are type Icell-surface glycoproteins with common structural features including anN-terminal immunoglobulin (IgV)-like domain with highly conservedcysteine residues, a threonine-rich mucin domain with O-linked andN-linked glycosylations, a single transmembrane domain, and acytoplasmic region. While the cytoplasmic domain of human TIM-1 andTIM-3 contain tyrosine phosphorylation motif(s), TIM-4 lacks aphosphotyrosine motif.

TIM-4 is expressed on myeloid cells, including dendritic cells (DCs) andmacrophages from spleen, lymph nodes, or peritoneal cavity. TIM-4 hasbeen demonstrated to bind to TIM-1, MerTK, Integrin αvβ3 andphosphatidyl serine via the IgV domain. MerTK and Integrin αvβ3 may playa role in certain TIM4 mediated biological activities (Nishi et al.(2014) Mol Cell Biol. 34(8):1512-20 and Toda et al. (2012) Mol CellBiol. 32(1):118-25). TIM-4 differentially regulates T cell homeostasisby inhibiting naïve T cells during the induction phase of an immuneresponse and enhancing T cell responses at the effector phase(Rodriguez-Manzanet et al., supra). For example, TIM-4 binds to TIM-1,which is present on activated T cells, and co-stimulates T cellproliferation.

TIM-4 binds to the phosphatidylserine (PS) through the FG-CC′ bindingcleft in the N-terminal immunoglobulin variable (IgV) domain. TIM-4 isupregulated following the release of DAMPs, and enhances efferocytosisof apoptotic cells by macrophages (Kobayashi, 2007; Albacker, et al., J.Immunol. 2010; 185:6839-6849; Mizui et al. Int. Immunol. 2008;20:695-708). In contrast, TIM-4 knockout mice have defects in lysosomaldegradation, antigen presentation and cross-priming (Miyanishi 2012,Rodrigues-Manzaneta, 2010).

TIM-4 has been demonstrated to be expressed on macrophages and DCs inthe tumor microenvironment (TME), and may modulate the interactionbetween myeloid cells and antigen-specific cytotoxic T cells withintumors. Bahgdadi et al. (supra) reported that apoptotic tumor cells wereingested by TIM-4+ bone-marrow derived macrophages (BMDMs), suggestingthat TIM-4 contributes to immune tolerance by promoting excessivelysosomal degradation of ingested tumor cells, leading to impaired tumorantigen presentation by TIM-4+ tumor-associated macrophages (TAMs).Inhibition of this function appears to promote antitumor activityfollowing monotherapy with an anti-TIM-4 blocking antibody anddemonstrates synergistic antitumor activity when combined with ananti-TIM-3 blocking antibody in the context of the vaccine therapy model(Baghadadi 2013).

The term “antagonist” as used with reference to TIM-4 refers to anymolecule that partially or fully inhibits one or more biologicalactivities of TIM-4, in vitro, in situ, or in vivo. Examples of suchbiological activities include binding of TIM-4 to PS, efferocytosis oftumor cells, and suppression of tumor antigen presentation, as well asthose further reported in the literature. TIM-4 antagonists may functionin a direct or indirect manner. For example, the TIM-4 antagonist mayfunction to partially or fully inhibit one or more biological activitiesof TIM-4, in vitro, in situ, or in vivo as a result of blocking directbinding to TIM-4 to PS and/or blocking the interaction of TIM-4 to otherPS receptors and reducing cellular ability to engage in efferocytosis oftumor cells. The antagonist may also function by interfering with TIM-4interaction with TIM-1 thereby preventing direct T cell regulatoryactivity. The TIM-4 antagonist may also function indirectly to partiallyor fully inhibit one or more biological activities of TIM-4, in vitro,in situ, or in vivo as a result of, e.g., inhibiting another moleculewhich then blocks TIM-4 activation or expression. It is contemplatedthat an antagonist may act as a molecule which functions indirectly toblock, inhibit or decrease TIM-4 expression or activity.

A TIM-4 antagonist may be any molecule that directly or indirectlyinhibits or decreases the activity of TIM-4 and reduces tumor growth,whether on its own or in combination with another treatment, such as aPD-1 antagonist. Exemplary TIM-4 antagonists include TIM-4 bindingscaffolds, such as anti-TIM-4 antibodies (“TIM-4 antibodies”), e.g.,chimeric, humanized or fully human antibodies, an antigen bindingportion thereof, or molecules that are based on or derived from any ofthese. TIM-4 antagonists may also be non-antibody proteins. For example,TIM-4 antagonist also include modified TIM-4 ligands or bindingproteins, e.g., TIM-1 and molecules that are derived from or based onPS. In addition, TIM-4-regulated biology may be interfered with byfusion proteins such as TIM-4: Ig.

A TIM-4 antagonist may be monovalent or multivalent. In certainembodiments, a TIM-4 antagonist is bivalent, trivalent, tetravalent, orbinds to 5, 6, 7, 8, 9, 10 or more TIM-4 epitopes, which may be the sameor different TIM-4 epitopes. In certain embodiments, a TIM-4 antagonistis a multivalent monospecific TIM-4 binding scaffold, e.g., a proteincomprising a TIM-4 binding scaffold that comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10 or more regions that specifically bind to the same TIM-4epitope, which binding regions may be composed of the same or adifferent amino acid sequence. For example, a TIM-4 antagonist may be aTIM-4 binding scaffold comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or morerepeats of the same TIM-4 binding region, e.g., the N-terminal IgVregion or portion thereof containing the FG-CC′ binding cleft of the IgVregion.

In certain embodiments, a TIM-4 antagonist binds specifically to TIM-4,but does not bind significantly or specifically to other members of theTIM family, such as TIM-1 or TIM-3. In other embodiments, a TIM-4antagonist binds specifically to TIM-4 and TIM-1.

In another embodiment, the TIM-4 antagonist is an antibody, e.g., anantibody that binds to human TIM-4 with a K_(D) of 10⁻⁷M, 5×10⁻⁸M,10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M or less, wherein the antibodyinhibits tumor growth, and/or increases tumor antigen specific CTLs.Antibodies binding to human TIM-4 are known in the art. Any of theseantibodies may be used in combination with a PD-1 antagonist, providedthat their combination results in inhibition of tumor growth orreduction in tumor size, e.g., in a subject having cancer. Exemplaryantibodies include those which bind specifically to the N-terminal IgVdomain of TIM-4 (e.g., the FG-CC″ binding cleft). Anti-TIM-4 antibodiesinclude those which bind human TIM-4, e.g., 9F4 (BioLegend). Anti-mouseanti-TIM4 antibodies include RMT4-53 (BioXCell, GeneTex), RMT4-54(BioLegend) F31-5G3 and 21H12 (BioLegend, BD Biosciences, respectively).Variants of these antibodies, such as antibodies comprising the CDRs ofthese antibodies, or antibodies that compete for binding to human ormurine TIM-4 with one of these antibodies or antibodies that bind to thesame or similar epitope on TIM-4 as one of these antibodies may be usedin combinations with a PD-1 antagonist.

In certain embodiments, an anti-TIM4 antibody for use in the methodsdescribed herein binds to a region on human TIM-4 that is not the IgVdomain, and can be, e.g., the stem domain or any other region in theextracellular domain of TIM-4, provided that the antibody antagonizesthe biological activity of TIM-4, and has at least an additive effectwith a PD-1 antagonist when used for treating cancer (relative to eitheragent alone).

In another embodiment, the TIM-4 antagonist is a multivalent agent, suchas a multimer (e.g., a polypeptide construct with trimerizing domain anda polypeptide that binds TIM-4).

Agents, which compete for binding to TIM-4 with any of the exemplaryagents listed herein, and which inhibit tumor growth or reduces tumorsize may also be used. Antibodies having VH and VL chains comprising anamino acid sequence that is at least 90%, 95%, 98% or 99% identical tothose of any of the anti-TIM-4 antibodies listed herein may be used.

Administration of a TIM-4 antagonist may be monitored by imaging with aphosphatidylserine (PS)-binding agent (e.g. annexin V peptides, TIM-4binding fragments), where the PS binding agent may be labeled forimaging, e.g. PET, SPECT, fluorescence, etc. The binding agent isbrought into contact with the target tumor cells, and the presence ofbound agent is indicative of PS being present and able to interact withTIM-4.

For example, Annexin V is a ubiquitous intracellular protein in humansthat has a nanomolar affinity for the membrane-bound constitutiveanionic phospholipid phosphatidylserine (PS), which is selectivelyexpressed on the surface of apoptotic or physiologically stressed cells.As such, radiolabeled forms of annexin V have been used in both animalmodels and human Phase I and Phase II trials for utilizing the tracer asan early surrogate marker of therapeutic efficacy (e.g., Blankenberg etal. Proc. Am. J. Thoracic. Soc. 2009; 6:469-476). For example, the invivo monitoring of PS within the tumor microenvironment may be performedwith anti-annexin V linked to a label radiotracer (for PET or SPECT), orwith a TIM-4 fusion protein linked to a label or radiotracer. Labelscould include q-dots for near IR imaging, or other more conventionallabels. pK/Kd studies may be performed with radiolabeled antibodies.

IV. Compositions

In one aspect, the present invention provides composition comprising aPD-1 antagonist and a TIM-4 antagonist (e.g., formulated together in asingle composition or separately formulated). In one embodiment, thePD-1 antagonist is nivolumab, pembrolizumab, durvalumab, atezolizumab,avelumab or PDR001. In one embodiment, the composition comprises a PD-1antagonist and a TIM-4 antagonist, wherein (a) the PD-1 antagonist is ananti-PD-1 antibody comprising the CDR1, CDR2 and CDR3 domains in a heavychain variable region having the sequence set forth in SEQ ID NO: 13,and the CDR1, CDR2 and CDR3 domains in a light chain variable regionhaving the sequence set forth in SEQ ID NO: 15; and (b) the TIM-4antagonist is an antibody. In another embodiment, the compositioncomprises a PD-1 antagonist and a TIM-4 antagonist, wherein (a) the PD-1antagonist is an anti-PD-L1 antibody comprising the CDR1, CDR2 and CDR3domains in a heavy chain variable region having the sequence set forthin SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a light chainvariable region having the sequence set forth in SEQ ID NO: 3 and (b)the TIM-4 antagonist is an antibody.

Pharmaceutical compositions suitable for administration to humanpatients are typically formulated for parenteral administration, e.g.,in a liquid carrier, or suitable for reconstitution into liquid solutionor suspension for intravenous administration.

In general, such compositions typically comprise a pharmaceuticallyacceptable carrier. As used herein, the term “pharmaceuticallyacceptable” means approved by a government regulatory agency or listedin the U.S. Pharmacopeia or another generally recognized pharmacopeiafor use in animals, particularly in humans. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil, glycerol polyethylene glycol ricinoleate, and the like. Water oraqueous solution saline and aqueous dextrose and glycerol solutions maybe employed as carriers, particularly for injectable solutions. Liquidcompositions for parenteral administration can be formulated foradministration by injection or continuous infusion. Routes ofadministration by injection or infusion include intravenous,intraperitoneal, intramuscular, intrathecal and subcutaneous.

For oral use, the pharmaceutical compositions of the present invention,may be administered, for example, in the form of tablets or capsules,powders, dispersible granules, or cachets, or as aqueous solutions orsuspensions. In the case of tablets for oral use, carriers which arecommonly used include lactose, corn starch, magnesium carbonate, talc,and sugar, and lubricating agents such as magnesium stearate arecommonly added. For oral administration in capsule form, useful carriersinclude lactose, corn starch, magnesium carbonate, talc, and sugar. Whenaqueous suspensions are used for oral administration, emulsifying and/orsuspending agents are commonly added.

In addition, sweetening and/or flavoring agents may be added to the oralcompositions. For intramuscular, intraperitoneal, subcutaneous andintravenous use, sterile solutions of the active ingredient(s) areusually employed, and the pH of the solutions should be suitablyadjusted and buffered. For intravenous use, the total concentration ofthe solute(s) should be controlled in order to render the preparationisotonic.

For preparing suppositories according to the invention, a low meltingwax such as a mixture of fatty acid glycerides or cocoa butter is firstmelted, and the active ingredient is dispersed homogeneously in the wax,for example by stirring. The molten homogeneous mixture is then pouredinto conveniently sized molds and allowed to cool and thereby solidify.

Liquid preparations include solutions, suspensions and emulsions. Suchpreparations are exemplified by water or water/propylene glycolsolutions for parenteral injection. Liquid preparations may also includesolutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas.

Also included are solid preparations which are intended for conversion,shortly before use, to liquid preparations for either oral or parenteraladministration. Such liquid forms include solutions, suspensions andemulsions.

V. Patient Populations

Provided herein are effective methods for treating cancer in a patient,e.g., using a combination of a TIM-4 antagonist and PD-1 antagonist. Inone embodiment, the patient suffers from a cancer selected from thegroup consisting of carcinoma, sarcoma, blastoma, leukemia and lymphoma.In another embodiment, the patient suffers from a cancer selected fromthe group consisting of non-small cell lung cancer, small cell lungcancer, gastrointenstinal cancer, colorectal cancer, stomach cancer,colon carcinoma and glioblastoma. In another embodiment, the patientsuffers from a cancer selected from the group consisting of chronicmyeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosomepositive acute lymphoblastic leukemia (Ph+ ALL), squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, glioma,gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer,endometrial cancer, kidney cancer, prostate cancer, thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervicalcancer, stomach cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer, gastric cancer, germ cell tumor,pediatric sarcoma, sinonasal natural killer, multiple myeloma, acutemyelogenous leukemia (AML), and chronic lymphocytic leukemia (CML).

VI. Additional Agents/Therapies

The combinations of the present invention (e.g., PD-1 antagonist incombination with TIM-4 antagonist) may also be used in conjunction withother well known therapies that are selected for their particularusefulness against the cancer that is being treated. Combinations of theinstant invention may alternatively be used sequentially with knownpharmaceutically acceptable agent(s) when inappropriate.

For example, the PD-1 antagonists and TIM-4 antagonist described hereincan further be used in combination (e.g., simultaneously or separately)with an additional treatment, such as irradiation, chemotherapy (e.g.,using camptothecin (CPT-11), 5-fluorouracil (5-FU), cisplatin,doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel,doxorubicin, 5-fu, or camptothecin+apo21/TRAIL (a 6× combo)), one ormore proteasome inhibitors (e.g., bortezomib or MG132), one or moreBcl-2 inhibitors (e.g., BH3I-2′ (bcl-xl inhibitor), AT-101(R-(−)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1)antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g.,smac7, smac4, small molecule smac mimetic, synthetic smac peptides (seeFulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), orAEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20antibodies (e.g., rituximab), angiogenesis inhibitors (e.g.,bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR, synthetictriterpenoids (see Hyer et al., Cancer Research 2005; 65:4799-808),c-FLIP (cellular FLICE-inhibitory protein) modulators (e.g., natural andsynthetic ligands of PPARγ (peroxisome proliferator-activated receptorγ), 5809354 or 5569100), kinase inhibitors (e.g., Sorafenib), and/orgenotoxic drugs.

The PD-1 antagonists and TIM-4 antagonists described herein can furtherbe used in combination with one or more anti-proliferative cytotoxicagents. Classes of compounds that may be used as anti-proliferativecytotoxic agents include, but are not limited to, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for use in the methods of theinvention, include, without limitation, taxanes, paclitaxel (paclitaxelis commercially available as TAXOL®), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone Bl, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D,cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with thechemotherapeutic methods of the invention, hormones and steroids(including synthetic analogs), such as 17a-Ethinylestradiol,Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,Dromostanolone propionate, Testolactone, Megestrolacetate,Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone,Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine,Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, ZOLADEX′,can also be administered to the patient. When employing the methods orcompositions of the present invention, other agents used in themodulation of tumor growth or metastasis in a clinical setting, such asantimimetics, can also be administered as desired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

A PD-1 antagonist and a TIM4 antagonist may also be combined with one ormore immunotherapy agent that stimulates the immune system, e.g., anagent that binds to human CTLA-4, LAG-3, GITR, OX40, DO, CSF-1R etc.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

VII. Treatment Protocols

Suitable treatment protocols for treating cancer in a patient include,for example, administering to the patient an effective amount of a PD-1antagonist (e.g., antibody) and a TIM-4 antagonist (e.g., antibody).

As used herein, adjunctive or combined administration(co-administration) includes simultaneous administration of the twoantagonists in the same or different dosage form, or separateadministration of the two antagonist (e.g., sequential administration).Thus, the PD-1 antagonist (e.g., antibody) and TIM-4 antagonist (e.g.,antibody) can be simultaneously administered in a single formulation.Alternatively, the PD-1 antagonist and TIM-4 antagonist can beformulated for separate administration and are administered concurrentlyor sequentially.

For example, the PD-1 antagonist can be administered first followed by(e.g., immediately followed by) the administration of the TIM-4antagonist, or vice versa. In one embodiment, the PD-1 antagonist isadministered prior to administration of the TIM-4 antagonist, e.g., thePD-1 antagonist is infused into the patient first, followed from 10minutes to 3 hours later by an infusion of the TIM-4 antagonist. In oneembodiment, the TIM-4 antagonist is administered prior to administrationof the PD-1 antagonist, e.g., the TIM-4 antagonist is infused into thepatient first, followed from 10 minutes to 3 hours later by an infusionof the PD-1 antagonist. Such concurrent or sequential administrationpreferably results in both antagonists being simultaneously present intreated patients. In another embodiment, the TIM-4 antagonist and thePD-1 antagonist are administered simultaneously.

In one embodiment, a subject is administered a single dose of a TIM-4antagonist and a single dose of the PD-1 antagonist, e.g., an anti-PD-1or anti-PD-L1 antibody. In certain embodiments, multiple (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10 or more) doses of a TIM-4 antagonist and multiple(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) doses of a PD-1 antagonistare administered to a subject in need of treatment. Administration ofthe TIM-4 antagonist and the PD-1 antagonist may be on the same day, oralternatively, the TIM-4 antagonist may be administered 1 or more daysbefore or after the PD-1 antagonist.

In one embodiment, administrations of a TIM-4 antagonist and a PD-1antagonist may be done weekly or monthly, in which regimen, they may beadministered on the same day (e.g., simultaneously), or one after theother (e.g., one or more minutes, hours or days before or after oneanother). In one embodiment, the TIM-4 antagonist and PD-1 antagonistare administered every three days.

In one embodiment, the dose of the PD-1 antagonist and/or TIM-4antagonist is varied over time. For example, the PD-1 antagonist and/orTIM-4 antagonist may be initially administered at a high dose and may belowered over time. In another embodiment, the PD-1 antagonist and/orTIM-4 antagonist is initially administered at a low dose and increasedover time.

In another embodiment, the amount of the PD-1 antagonist and/or TIM-4antagonist administered is constant for each dose. In anotherembodiment, the amount of the PD-1 antagonist and/or TIM-4 antagonistvaries with each dose. For example, the maintenance (or follow-on) doseof the antagonist can be higher or the same as the loading dose which isfirst administered. In another embodiment, the maintenance dose of theantagonist a can be lower or the same as the loading dose. A clinicianmay utilize preferred dosages as warranted by the condition of thepatient being treated. The dose may depend upon a number of factors,including stage of disease, etc. The specific dose that should beadministered based upon the presence of one or more of such factors iswithin the skill of the artisan. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired. Intermittent therapy (e.g., one week out of three weeks orthree out of four weeks) may also be used.

In one embodiment, the TIM-4 antagonist (e.g., antibody) is administeredat a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg bodyweight. In another embodiment, the PD-1 antagonist (e.g., antibody) isadministered at a dose of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kgbody weight. Generally, 200 μg/mouse is approximately 10 mg/kg and 100μg/mouse is approximately 5 mg/kg. Therefore, based on the experimentsdescribed herein, one or more doses of 1-20 mg/kg body weight, 1-10mg/kg body weight, 5-20 mg/kg body weight or 5-10 mg/kg body weight of aTIM-4 antagonist and PD-1 antagonist may be administered to a subject.In certain embodiments, a dose of 0.3 mg/kg to 10 mg/kg body weight of aTIM-4 antagonist is used and a dose of at least 1 mg/kg, e.g., 1-10mg/kg body weight of a PD-1 antagonist is used.

VIII. Outcomes

Patients, e.g., humans, treated according to the methods disclosedherein preferably experience improvement in at least one sign of cancer.In one embodiment, improvement is measured by a reduction in thequantity and/or size of measurable tumor lesions. In another embodiment,lesions can be measured on chest x-rays or CT or MRI films. In anotherembodiment, cytology or histology can be used to evaluate responsivenessto a therapy.

In one embodiment, the patient treated exhibits a reduction in size of atumor, reduction in number of metastasic lesions over time, completeresponse, partial response, and stable disease. In another embodiment,the patient treated experiences tumor shrinkage and/or decrease ingrowth rate, i.e., suppression of tumor growth. In another embodiment,unwanted cell proliferation is reduced or inhibited. In yet anotherembodiment, one or more of the following can occur: the number of cancercells can be reduced; tumor size can be reduced; cancer cellinfiltration into peripheral organs can be inhibited, retarded, slowed,or stopped; tumor metastasis can be slowed or inhibited; tumor growthcan be inhibited; recurrence of tumor can be prevented or delayed; oneor more of the symptoms associated with cancer can be relieved to someextent.

In another embodiment, the methods of treatment produce a comparableclinical benefit rate (CBR=CR (complete response), PR (partial response)or SD (stable disease)≥6 months) better than that achieved by a PD-1(e.g., antibody) or TIM-4 antagonist (e.g., antibody) alone. In otherembodiments, the improvement of clinical benefit rate is about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80% or more, e.g., compared to treatment with aPD-1 antagonist or TIM-4 antagonist alone or relative to tumor growth onthe first day of treatment or immediately before initiation oftreatment.

In another embodiment, administration of a PD-1 antagonist and a TIM-4antagonist results in at least a three-fold reduction (e.g., a 3.5-foldreduction) in tumor volume, e.g., relative to treatment with the PD-1antagonist or the TIM-4 antagonist alone or relative to tumor growth onthe first day of treatment or immediately before initiation oftreatment.

In a further embodiment, administration of a PD-1 antagonist and a TIM-4antagonist results in tumor growth inhibition of at least 80%, e.g.,relative to treatment with the PD-1 antagonist or TIM-4 antagonist aloneor relative to tumor growth on the first day of treatment or immediatelybefore initiation of treatment.

In certain embodiments, administration of a PD-1 antagonist and a TIM-4antagonist reduces tumor mass by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99% relative to the tumor mass prior to initiationof the treatment or on the first day of treatment.

In some embodiment, the tumor mass is no longer detectable followingtreatment as described herein. In some embodiments, a subject is inpartial or full remission. In certain embodiments, a subject has anincreased overall survival, median survival rate, and/or progressionfree survival.

IX. Kits and Unit Dosage Forms

Also provided herein are kits which include a pharmaceutical compositioncontaining (a) a PD-1 antagonist and (b) a TIM-4 antagonist and apharmaceutically-acceptable carrier, in a therapeutically effectiveamount adapted for use in the preceding methods. In one embodiment, thePD-1 antagonist is an antibody (e.g., 5C4 or 12A4, respectively). Inanother embodiment, the TIM-4 antagonist is an antibody. The kitsoptionally also can include instructions, e.g., comprisingadministration schedules, to allow a practitioner (e.g., a physician,nurse, or patient) to administer the composition contained therein to apatient having cancer. The kit also can include a syringe.

Optionally, the kits include multiple packages of the single-dosepharmaceutical compositions each containing an effective amount of thePD-1 antagonist and the TIM-4 antagonist for a single administration inaccordance with the methods provided above. Instruments or devicesnecessary for administering the pharmaceutical composition(s) also maybe included in the kits. For instance, a kit may provide one or morepre-filled syringes containing an amount of the PD-1 antagonist and theTIM-4 antagonist.

In one embodiment, the present invention provides a kit for treatingcancer in a patient, the kit comprising:

(a) a dose of a PD-1 antagonist;

(b) a dose of a TIM-4 antagonist; and

(c) instructions for using the PD-1 antagonist and TIM-4 antagonist inthe methods described herein.

In one embodiment, the present invention provides a kit for treatingcancer in a patient, the kit comprising:

(a) one or more doses of a PD-1 antagonist;

(b) one or more doses of a TIM-4 antagonist; and

(c) instructions for using the PD-1 antagonist and TIM-4 antagonist inthe methods described herein.

In certain embodiments, the TIM-4 antagonist is an antibody. In certainembodiments, the PD-1 antagonist is an antibody. In particularembodiments, the PD-1 antagonist is an anti-PD-1 antibody comprising theCDR1, CDR2 and CDR3 domains in a heavy chain variable region having thesequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domainsin a light chain variable region having the sequence set forth in SEQ IDNO: 15. In another particular embodiment, the PD-1 antagonist is ananti-PD-L1 antibody comprising antibody comprises the CDR1, CDR2 andCDR3 domains in a heavy chain variable region having the sequence setforth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a lightchain variable region having the sequence set forth in SEQ ID NO: 3.

The following examples are merely illustrative and should not beconstrued as limiting the scope of this disclosure in any way as manyvariations and equivalents will become apparent to those skilled in theart upon reading the present disclosure.

The contents of all references, Genbank entries, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Materials and Methods

Animals

Ten to eleven-week-old female C57/BL6 mice (Harlan) were used in thestudies. Mice received food and water ad libitum and were maintained ina controlled environment according to Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC) Internationalregulations. All animal studies have been approved by the appropriateethics committee and have therefore been performed in accordance withthe ethical standards laid down in the 1964 Declaration of Helsinki andits later amendments.

Antibodies

Anti-mouse PD-1 mAb (anti-mPD-1 mAb) clone 4H2, mouse IgG1 isotype wasproduced and purified by Bristol-Myers Squibb (Biologics Discovery, CA).The anti-TIM-4 mAb, RMT4-53 has been previously described (Yeung et al.,J. Immunol. 2009; 191:4447-4455). Briefly, Sprague Dawley rats wereimmunized with a TIM-4-Ig fusion protein containing the extracellulardomain of murine TIM-4 (aa 1-288) to the Fc portion of mouse IgG2a, andfusing LN cells with P3U1 myeloma cells. RMT4-53 reacts with TIM-4/NRKcells but not with parental NRK or other transfected TIM family (TIM-1B6/NRK, TIM-1 BALB/NRK, TIM-2/NRK, TIM-3 B6/NRK, TIM-3 BALB/NRK) cells.Antibodies were certified to have <0.5 EU/mg endotoxin levels, >95%purity and <5% high molecular weight species. Stock solutions ofanti-mPD-1 mAb (clone 4H2; mouse IgG1) and anti-mTIM-4 antibody (cloneRM 4-53; rat IgG2a), were kept at 4° C. prior to use. Dosing solutionsof anti-mPD-1 mAb and anti-mTIM-4 mAb were prepared in sterile phosphatebuffered saline (pH 7.0) and maintained at 4° C.

Cell Lines

CT-26 is an undifferentiated colon carcinoma cell line with a fibroblastmorphology (ATCC). Mice inoculated subcutaneously develop lethal tumorsat 80% frequency with 10³ cells and at 100% with 10⁴ cells. Pulmonarymetastases develop when mice are inoculated, intravenously, with 10⁴cells (Wang et al. J. Immunol. 1995; 154:4685-4692). The MC38 cell linewas derived from C57BL6 murine colon adenocarcinoma cells (CD44+ALDH1+)with an epithelial morphology.

Expression of TIM-4

Expression of TIM-4 on mouse peritoneal macrophages and TAMs wasconfirmed by flow cytometry.

Immunohistochemistry

Immunohistochemical analysis using Rabbit anti-TIM4 (Atlas Antibodies,HPA015625) demonstrated expression of TIM-4 on tissue residentmacrophages present in human tonsil, lung and liver. Expression wasdemonstrated on fixed tissue was sectioned and applied to SuperfrostPlus glass slides at a thickness of 5 microns. Sections were allowed todry overnight at room temperature. Tissues were then deparaffinized inxylene followed by hydration to distilled water in graded series ofethanol. Antigen Retrieval was performed using HIER with Biocare MedicalEDTA, pH 8.2 (1×) at approximately 115° C. for 1 minute and then cooledfor 20 minutes at RT. Sections were rinsed in TNT Buffer (TBS+0.5%Tween) and isolated using a PAP pen. Blocking was performed with BiocareMedical Background Sniper for 30 minutes at RT, followed by 1×TNT Bufferrinse, incubation in Dako Peroxidase Block for 10 minutes at RT and2×TNT Buffer rinses. Primary Antibody staining was performed using a1/1000 dilution in Biocare Medical Renaissance antibody diluent andbackground reducer for 60 minutes at RT and 3×TNT Buffer rinses.Detection antibody was applied using appropriate number of drops tocover section with Biocare Medical MACH3 Rabbit polymer-HRP andincubating for 30 minutes at RT followed by and 3×TNT Buffer rinses.Samples were incubated with Biocare Medical Betazoid DAB and reactionstopped by placing slides in water). Sections were counterstained inHematoxylin, Leica for 30 seconds, washed in tap water, incubated inBluing Solution for 45 seconds to 1 minute, washed 2× in water,dehydrated in graded series of alcohol, and Xylene and then mounted.

Example 1: Inhibition of Efferocytosis by Anti-TIM-4 Antibody

Thymocytes were isolated from BALB/c mice and cultured with or withoutdexamethasone for 4 hours to induce apoptosis. Cells were then incubatedwith pHrodo dye for 5-10 minutes according to manufacturer'sinstructions (Thermofisher Scientific). Briefly, 2×10⁷ cells were washedin media without serum 1× and resuspended in 1 mL of Diluent C (CatalogNumber G8278). Cells were then stained by adding 1 mL of 2× dye solution(by adding 4 mL of the PKH26 ethanolic dye solution (Catalog NumberP9691) to 1 mL of Diluent C) and mixing sample by pipetting. Incubationwas for 5 minutes with periodic mixing. Staining reaction was stopped byadding an equal volume of serum and incubating 1 minute. Cells were thenwashed 1× in media without serum and 2× in PBS.3×10⁶ pHrodo-labeledcells were injected i.p. into BALB/c mice and peritoneal macrophageswere collected by lavage by injecting 3-5 mL of ice cold PBS+2 mM EDTAinto the peritoneal cavity of euthanized mice, palpating the abdomen andthen collecting the solution via needle aspiration. Experimental animalswere injected with RMT4-53 anti-TIM4 antibody 1 hour prior to injectionof the pHrodo-labeled thymocytes T.

The uptake of apoptotic cells by peritoneal macrophages was measuredusing flow cytometry. Briefly, peritoneal macrophages were washed 2× inPBS, resuspended in 100 viability dye (eBioscience) and incubated on ice15 minutes. Cells were washed 1× in PBS and incubated in FcBlock(BioLegend) for 15 minutes on ice. Cells were then stained withantibodies to CD45 (BD), Tim-4 (BioLegend), CD 11b (BioLegend), CD 206(BioLegend), and F4/80+(BioLegend), for 30 minutes on ice. Cells werethen washed 1× and resuspended in Perm/Fix. Samples were then collectedon a LSR Fortessa X20 (BD). To identify cells that engaged inefferocytosis, CD45+/CD11b+ myeloid cells from the live cell populationwere gated on. Out of these cells, macrophages that engaged inefferocytosis were defined as F4/80+, OPKH26+. The results are set forthin Table 2.

TABLE 2 % Apoptotic Cells Control 25-40 Control + Dex 50-70Experimental + anti-TIM4 10-15 Experimental + Dex and TIM-4 22-42The results demonstrate an increase in efferocytosis of apoptotic cells(50-70%) relative to non-apoptotic cells (25-40%). The addition ofanti-TIM-4 blocking anybody greatly reduced uptake of both viable andapoptotic cells. This demonstrates that TIM-4 is an important mediatorof cellular uptake.

Example 2: Inhibition of Tumor Growth In Vivo in the CT26 Model byCombination Treatment with Anti-TIM-4 Antibody and Anti-PD-1 Antibody

An experiment was conducted in a murine tumor model to test thehypothesis that the combination of anti-TIM-4 and anti-PD-1 wouldpotentiate anti-tumor efficacy. SC CT-26 mice, a colon adenocarcinomatumor model (TGM-1438), were evaluated for tumor growth after treatmentwith the anti-TIM4 antibody, RTM-453, alone or in combination with ananti-PD1 antibody, IgG1 D265A.

Female BALB/c mice (Harlan; approximately 8-9 wk old) weresubcutaneously implanted with 10⁶ CT-26 cells on day 0. Sixty mice wereevaluated by body weight and tumor measurements after 2×/wk dosingaccording to the following schedule:

TABLE 3 Dosing Schedule: Group N Treatment Dosing Regimen 1 15 mIgG1isotype control mAb 0.45 mg, IP, d. 7, 10, 13 2 15 mPD-1 IgG1 D265AmAb + 0.2 + 0.25* mg, IP, d. 7, Control mAb 10, 13 4 15 mTIM-4 rat IgG2amAb + 0.25 + 0.2* mg, IP, d. 7, Control mAb 10, 13 6 15 mTIM-4 + mPD-1mAbs 0.25 mg + 0.2 mg, IP, d. 7, 10, 13

On the days of dosing, the two antibodies were combined and a total of1004, of combined antibody was injected into the mice. Spleens andtumors from five mice per group were collected and processed for flowcytometry analysis of tumor infiltrating lymphocytes (TILs), and spleenimmune cells on day 16 according to the following protocol:

Briefly, cells were resuspended in PBS and aliquoted into plates(spleens 2×10⁶/well, all tumor cells/well) and then were washed 2× inPBS, backspace. Cells were then resuspended in 100 μL viability dye(eBioscience) and incubated on ice 15 minutes. Cells were washed 1× inPBS and incubated in 50 μL FcBlock (BioLegend) for 15 minutes on ice andthen stained with 50 μL of antibody stain mix as described below for 30minutes. Cells were then washed 1× with FACS buffer and resuspended inPerm/Fix. Samples were then collected on a LSR Fortessa X20 (BD).

T Cell Panel

Fluor Specificity Clone Antibody Cat# Company Ab diln AlexaFluor 488CD107a 1D4B Alexa Fluor ® 488 anti-mouse 121608 BioLegend 1:100 (FITC)CD107a (LAMP-1) Antibody PE CD115 AFS98 PE anti-mouse CD115 (CSF-1R)135506 BioLegend 1:100 Antibody PerCP-Cy5.5 CD49b DX5 PerCp/Cy5.5anti-mouse CD49b 108916 BioLegend 1:100 (pan-NK cells) antibody BUV 395CD45 30-F11 BUV395 rat anti-mouse CD45 564279 eBioscience 1:100 antibodyBV 421 CD8 53-6.7 Brilliant Violet 421 ™ anti-mouse 100738 BioLegend1:200 CD8a antibody BV 510 CD4 RM4-5 Brilliant Violet 510 ™ anti-mouse100553 BioLegend 1:200 CD4 antibody BV 605 Thy1.2 53-2.1 BrilliantViolet 605 ™ anti-mouse 140317 BioLegend 1:200 CD90.2 (Thy-1.2) antibodyBV 711 CD62L MEL-14 Brilliant Violet 711 ™ anti-mouse 104445 BioLegend1:200 CD62L antibody BV 785 CD44 IM7 Brilliant Violet 785 ™ anti-mouse103041 BioLegend 1:200 CD44 antibody APC FoxP3 FJK-16s Anti-Mouse/RatFoxp3 APC 17-5773-80B eBioscience 1:100 PE-Cy7 Ki-67 SolA15Anti-Mouse/Rat Ki-67 PE-Cyanine7 25-5698-82 eBioscience 1:100 APC eFluor780 Fixable n/a eFluor ® 780 Fixable Viability Dye 65-0865-14eBioscience 1:1000 (APC-Cy7) Viability Dye FC Block block 93 TruStainFcX anti-mouse CD16/32 101320 BioLegend

-   -   Ki-67+ to assess proliferating cells;    -   CD44/CD62L analysis to determine subset T cells into naïve,        activated, and memory T cell subsets;    -   FoxP3 to assess population of Tregs (FoxP3+CD4+ T cells);    -   CD8+CD107a+ to assess degranulating CD8+ T cells        (Antigen/Tumor-specific CTL)

Myeloid Panel

Fluor Specificity Clone Antibody Cat# Company AlexaFluor 488 CD11c N418FITC anti-mouse CD11c 117306 BioLegend 1:100 (FITC) Antibody PE CD115AFS98 PE anti-mouse CD115 (CSF-1R) 135506 BioLegend 1:100 AntibodyPerCP-Cy5.5 CD49b DX5 PerCp/Cy5.5 anti-mouse CD49b 108916 BioLegend1:100 (pan-NK cells) antibody BUV 395 CD45 30-F11 BUV395 rat anti-mouseCD45 564279 eBioscience 1:100 antibody BV 421 Ly6C HK1.4 BrilliantViolet 421 ™ anti- 128032 BioLegend 1:200 mouse Ly6C antibody BV 605 Thy1.2 53-2.1 Brilliant Violet 605 ™ anti- 140317 BioLegend 1:200 (CD90.2)mouse Thy-1.2 antibody BV 711 Gr-1 RB6-8C5 Brilliant Violet 711 ™ anti-104731 BioLegend 1:200 mouse GR-1 antibody BV 785 F4-80 BM8 BrilliantViolet 785 ™ anti- 123141 BioLegend 1:200 mouse F4/80 antibody APC Ly6G1A8 APC anti-mouse Ly6G antibody 127614 BioLegend 1:200 AF-700 CD11bM1/70 Alexa Fluor 700 anti- 101222 BioLegend 1:100 mouse/human CD11bantibody PE-Cy7 MHCII M5/114.15.2 PE-Cy7 anti-mouse I-A/I-E 107630eBioscience 1:100 antibody APC eFluor 780 Fixable n/a eFluor ® 780Fixable Viability 65-0865-14 eBioscience 1:1000 (APC-Cy7) Viability DyeDye FC Block block 93 TruStain FcX anti-mouse 101320 BioLegend CD16/32

-   -   Myeloid Cell subsetting for TAMs, MDSCs, NK cells, B cells    -   CD80, CD86 staining for activated APCs

The myeloid populations in spleen and tumors are shown in FIG. 3. TheCD8+ T cell populations in spleen and tumor infiltrating lymphocytes areshown in FIG. 4. The CD4+ cell populations in spleen and tumorinfiltrating lymphocytes are shown in FIG. 5. The data demonstrate ashift in myeloid cell populations, and an increase in activated T cellsin tumor infiltrating lymphocytes.

The remaining ten mice per group were monitored for tumor growth. Tumorsize and body weights were measured twice weekly. Tumor size (measuredas mm³) was calculated by multiplying the tumor length by the square ofthe tumor width divided by 2. Treatments were initiated whensubcutaneous tumors reached a median size of 200 mm³ (establishedmodel).

Anti-TIM-4 antibody administered in combination with anti-PD-1 antibodyprovided enhanced anti-tumor activity above the activity of either agentalone. The combination demonstrated decreased tumor growth rates (FIGS.6-8), and an increased overall survival rate (FIG. 9). In sum, thecombination of the TIM-4 mAb and the PD-1 mAb resulted in synergisticactivity compared to the activity elicited by single agents alone.Therefore, results from this study demonstrate that a combinationregimen of anti-mTIM-4 mAb and PD-1 mAb is well-tolerated and result inmarked antitumor activity.

Example 3: Inhibition of Tumor Growth In Vivo in the MC38 Model byCombination Treatment with Anti-TIM-4 Antibody and Anti-PD-1 Antibody

MC38 mice, a colon adenocarcinoma tumor model, were evaluated for tumorgrowth after treatment with the anti-TIM4 antibody, RTM-453, alone or incombination with an anti-PD1 antibody, IgG1 D265A.

Female C57/131,6 mice (Harlan; approximately 8-9 wk old) weresubcutaneously implanted with 10⁶ MC38 cells on day 0. Mice were dosedbeginning on Day 6. Three doses at 200 μg/injection were administered IPevery 4th day. When the two antibodies were administered to a mouse, theantibodies were first combined, and administered together into themouse. Tumors were measured using calipers and tumor volumes calculatedusing the formula (L2×W)/2. Progression Free Survival defined as # ofdays for tumor to reach 4× initial tumor volume.

The results are provided in FIGS. 10, 11 and 12, and Table X. FIG. 10,which provides the percentage of progression free survival relative tothe days post implant, show that administration of the combination ofthe anti-PD-1 and anti-TIM4 antibodies results in a higher percentage ofprogression free survival relative to each antibody separately andrelative to the isotype control. Table X shows that the combinationtreatment provided more complete regressions relative to each antibodyseparately and relative to the isotype control. FIG. 11, which shows theMC38 mean tumor volume relative to the days post implant, indicates thatthe combination treatment reduced the mean tumor volume more relative toeach antibody separately and relative to the isotype control. FIG. 12A-Cshows the MC38 individual tumor volumes, and confirms the results shownin the FIGS. 10, 11 and Table X.

TABLE 5 Number of mice with complete regressions of tumors CompleteTreatment Regressions Isotype Control 0/10 Anti-PD1 Alone 2/10 anti-TIM4(RMT4-53 mIgG1 D265A) Alone 0/10 Anti-PD1 + anti-TIM4 (RMT4-53 mIgG1D265A) 4/10

Thus, administration of a combination of an anti-TIM4 antibody with ananti-PD-1 antibody in the CT26 and MC39 animal models resulted in astronger anti-tumor effect relative to each antibody alone.

SUMMARY OF THE SEQUENCE LISTING SEQ ID NO: SEQUENCE  1Heavy Chain Variable Region (VH) Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVS GSPFGMDVWGQGTTVTVSS 2 Heavy Chain Variable Region (VH) Nucleotide SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743)cag gtc cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg tcctcg gtg aag gtc tcc tgc aag act tct gga gac acc ttc agc acc tatgct atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atggga ggg atc atc cct ata ttt ggt aaa gca cac tac gca cag aag ttccag ggc aga gtc acg att acc gcg gac gaa tcc acg agc aca gcc tacatg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat ttt tgtgcg aga aag ttt cac ttt gtt tcg ggg agc ccc ttc ggt atg gac gtctgg ggc caa ggg acc acg gtc acc gtc tcc  3Light Chain Variable Region (VL) Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFG QGTKVEIK  4Light Chain Variable Region (VL) Nucleotide SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743)gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggggaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tactta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atctat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggcagt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cctgaa gat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg ccg acgttc ggc caa ggg acc aag gtg gaa atc aaa  5Heavy Chain CDR1 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) TYAIS  6 Heavy Chain CDR2 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) GIIPIFGKAHYAQKFQ  7 Heavy Chain CDR3 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) KFHFVSGSPFGMDV  8 Light Chain CDR1 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) RASQSVSSYLA  9 Light Chain CDR2 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) DASNRAT 10 Light Chain CDR3 Amino Acid SequenceAnti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.7,943,743) QQRSNWPT 11 Heavy Chain Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(variable region underlined; constant region bold)QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDT AVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 12 Light Chain Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(variable region underlined; constant region bold)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPR TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC13 Heavy Chain Variable Region (VH) Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 4 from WO 2006/121168)QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATND DYWGQGTLVTVSS 14Heavy Chain Variable Region (VH) Nucleotide SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 60 from WO 2006/121168)cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg tccctg aga ctc gac tgt aaa gcg tct gga atc acc ttc agt aac tct ggc atgcac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg gca gtt atttgg tat gat gga agt aaa aga tac tat gca gac tcc gtg aag ggc cga ttcacc atc tcc aga gac aat tcc aag aac acg ctg ttt ctg caa atg aacagc ctg aga gcc gag gac acg gct gtg tat tac tgt gcg acaaac gac gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca 15Light Chain Variable Region (VL) Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 11 from WO 2006/121168)EIVLTOSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQ GTKVEIK 16Light Chain Variable Region (VL) Nucleotide SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 67 from WO 2006/121168)gaa att gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg gaaaga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agt agt tac tta gcctgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gcatcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct gggaca gac ttc act ctc acc atc agc agc cta gag cct gaa gat ttt gca gtttat tac tgt cag cag agt agc aac tgg cct cgg acg ttcggc caa ggg acc aag gtg gaa atc aaa 17Heavy Chain CDR1 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 18 from WO 2006/121168) NSGMH 18Heavy Chain CDR2 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 25 from WO 2006/121168) VIWYDGSKRYYADSVKG 19Heavy Chain CDR3 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 32 from WO 2006/121168) NDDY 20Light Chain CDR1 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 39 from WO 2006/121168) RASQSVSSYLA 21Light Chain CDR2 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 46 from WO 2006/121168) DASNRAT 22Light Chain CDR3 Amino Acid SequenceAnti-PD-1 mAb (5C4 in WO 2006/121168)(SEQ ID NO: 53 from WO 2006/121168) QQSSNWPRT 23Complete PD-1 sequence (GenBank Accession No.: U64863)agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct ggtggggctgctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgggctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc cccaccttcttcccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc agcttctccaacacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac cagacggacaagctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc ttccgtgtcacacaactgcc caacgggcgt gacttccaca tgagcgtggt cagggcccgg cgcaatgacagcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc aaagagagcctgcgggcaga gctcagggtg acagagagaa gggcagaagt gcccacagcc caccccagcccctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg ggcggcctgctgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg gccgcacgagggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctgtgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc ccggagccccccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct agcggaatgggcacctcatc ccccgcccgc aggggctcag ccgacggccc tcggagtgcc cagccactgaggcctgagga tggacactgc tcttggcccc tctgaccggc ttccttggcc accagtgttctgcagaccct ccaccatgag cccgggtcag cgcatttcct caggagaagc aggcagggtgcaggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg ctccagcctgcacctgcacc aggcacagcc ccaccacagg actcatgtct caatgcccac agtgagcccaggcagcaggt gtcaccgtcc cctacaggga gggccagatg cagtcactgc ttcaggtcctgccagcacag agctgcctgc gtccagctcc ctgaatctct gctgctgctg ctgctgctgctgctgctgcc tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc cccggagcctcctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt gcccctggcagtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag tgggaggtacatggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga agtttcagggaaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctccc acctttacacatgcccaggc agcacctcag gccctttgtg gggcagggaa gctgaggcag taagcgggcaggcagagctg gaggcctttc aggccagcca gcactctggc ctcctgccgc cgcattccaccccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc aggagggcagggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg agggcagaagtgcaggcacc tagggccccc catgtgccca ccctgggagc tctccttgga acccattcctgaaattattt aaaggggttg gccgggctcc caccagggcc tgggtgggaa ggtacaggcgttcccccggg gcctagtacc cccgcgtggc ctatccactc ctcacatcca cacactgcacccccactcct ggggcagggc caccagcatc caggcggcca gcaggcacct gagtggctgggacaagggat cccccttccc tgtggttcta ttatattata attataatta aatatgagag catgct24 Human PD-L1 amino acid sequence - isoform a precursor (GenBankAccession No. NP_054862.1)MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEMEDKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTTTTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET 25Human PD-L1 amino acid sequence - isoform b precursor (GenBankAccession No. NP_001254635.1)MRIFAVFIFM TYWHLLNAPY NKINQRILVV DPVTSEHELT CQAEGYPKAE VIWTSSDHQVLSGKTTTTNS KREEKLFNVT STLRINTTTN EIFYCTFRRL DPEENHTAEL VIPELPLAHPPNERTHLVIL GAILLCLGVA LTFIFRLRKG RMMDVKKCGI QDTNSKKQSD THLEET 26Human TIM-4 amino acid sequence - isoform 1(GenBank Accession No. NP_612388.2)MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTTTSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDSWSSVESTSADTVLLTSKESKVWDLPSTSHVSMWKTSDSVSSPQPGASDTAVPEQNKTTKTGQMDGIPMSMKNEMPISQLLMIIAPSLGFVLFALFVAFLLRGKLMETYCSQKHTRLDYIGDSKNVLNDVQHGREDEDGLFTL 27Homo sapiens T-cell (TIMD4), transcript variant 1, mRNA(GenBank Accession No. NM_138379.2)ATAAGAGGTTGGGCTTTGGATAGATAGACAGACTCCTGGGTCCGGTCAACCGTCAAAATGTCCAAAGAACCTCTCATTCTCTGGCTGATGATTGAGTTTTGGTGGCTTTACCTGACACCAGTCACTTCAGAGACTGTTGTGACGGAGGTTTTGGGTCACCGGGTGACTTTGCCCTGTCTGTACTCATCCTGGTCTCACAACAGCAACAGCATGTGCTGGGGGAAAGACCAGTGCCCCTACTCCGGTTGCAAGGAGGCGCTCATCCGCACTGATGGAATGAGGGTGACCTCAAGAAAGTCAGCAAAATATAGACTTCAGGGGACTATCCCGAGAGGTGATGTCTCCTTGACCATCTTAAACCCCAGTGAAAGTGACAGCGGTGTGTACTGCTGCCGCATAGAAGTGCCTGGCTGGTTCAACGATGTAAAGATAAACGTGCGCCTGAATCTACAGAGAGCCTCAACAACCACGCACAGAACAGCAACCACCACCACACGCAGAACAACAACAACAAGCCCCACCACCACCCGACAAATGACAACAACCCCAGCTGCACTTCCAACAACAGTCGTGACCACACCCGATCTCACAACCGGAACACCACTCCAGATGACAACCATTGCCGTCTTCACAACAGCAAACACGTGCCTTTCACTAACCCCAAGCACCCTTCCGGAGGAAGCCACAGGTCTTCTGACTCCCGAGCCTTCTAAGGAAGGGCCCATCCTCACTGCAGAATCAGAAACTGTCCTCCCCAGTGATTCCTGGAGTAGTGTTGAGTCTACTTCTGCTGACACTGTCCTGCTGACATCCAAAGAGTCCAAAGTTTGGGATCTCCCATCAACATCCCACGTGTCAATGTGGAAAACGAGTGATTCTGTGTCTTCTCCTCAGCCTGGAGCATCTGATACAGCAGTTCCTGAGCAGAACAAAACAACAAAAACAGGACAGATGGATGGAATACCCATGTCAATGAAGAATGAAATGCCCATCTCCCAACTACTGATGATCATCGCCCCCTCCTTGGGATTTGTGCTCTTCGCATTGTTTGTGGCGTTTCTCCTGAGAGGGAAACTCATGGAAACCTATTGTTCGCAGAAACACACAAGGCTAGACTACATTGGAGATAGTAAAAATGTCCTCAATGACGTGCAGCATGGAAGGGAAGACGAAGACGGCCTTTTTACCCTCTAACAACGCAGTAGCATGTTAGATTGAGGATGGGGGCATGACACTCCAGTGTCAAAATAAGTCTTAGTAGATTTCCTTGTTTCATAAAAAAGACTCACTTATTCCATGGATGTCATTGATCCAGGCTTGCTTTAGTTTCATGAATGAAGGGTACTTTAGAGACCACAACTTCTCTGTCAAAAAHuman TIM-4 amino acid sequence isoform 2(GenBank Accession No. NP_001140198.1)MSKEPLILWLMIEFWWLYLTPVTSETVVTEVLGHRVTLPCLYSSWSHNSNSMCWGKDQCPYSGCKEALIRTDGMRVTSRKSAKYRLQGTIPRGDVSLTILNPSESDSGVYCCRIEVPGWFNDVKINVRLNLQRASTTTHRTATTTTRRTTTTSPTTTRQMTTTPAALPTTVVTTPDLTTGTPLQMTTIAVFTTANTCLSLTPSTLPEEATGLLTPEPSKEGPILTAESETVLPSDSWSSVESTSADTVLLTSKASDTAVPEQNKTTKTGQMDGIPMSMKNEMPISQLLMIIAPSLGFVLFALFVAFLLRGKLMETYCSQKHTRLDYIGDSKNVLNDVQHGREDEDGLFTLHomo sapiens T-cell (TIMD4), transcript variant 2, mRNA(GenBank Accession No. NM_001146726.1)ATAAGAGGTTGGGCTTTGGATAGATAGACAGACTCCTGGGTCCGGTCAACCGTCAAAATGTCCAAAGAACCTCTCATTCTCTGGCTGATGATTGAGTTTTGGTGGCTTTACCTGACACCAGTCACTTCAGAGACTGTTGTGACGGAGGTTTTGGGTCACCGGGTGACTTTGCCCTGTCTGTACTCATCCTGGTCTCACAACAGCAACAGCATGTGCTGGGGGAAAGACCAGTGCCCCTACTCCGGTTGCAAGGAGGCGCTCATCCGCACTGATGGAATGAGGGTGACCTCAAGAAAGTCAGCAAAATATAGACTTCAGGGGACTATCCCGAGAGGTGATGTCTCCTTGACCATCTTAAACCCCAGTGAAAGTGACAGCGGTGTGTACTGCTGCCGCATAGAAGTGCCTGGCTGGTTCAACGATGTAAAGATAAACGTGCGCCTGAATCTACAGAGAGCCTCAACAACCACGCACAGAACAGCAACCACCACCACACGCAGAACAACAACAACAAGCCCCACCACCACCCGACAAATGACAACAACCCCAGCTGCACTTCCAACAACAGTCGTGACCACACCCGATCTCACAACCGGAACACCACTCCAGATGACAACCATTGCCGTCTTCACAACAGCAAACACGTGCCTTTCACTAACCCCAAGCACCCTTCCGGAGGAAGCCACAGGTCTTCTGACTCCCGAGCCTTCTAAGGAAGGGCCCATCCTCACTGCAGAATCAGAAACTGTCCTCCCCAGTGATTCCTGGAGTAGTGTTGAGTCTACTTCTGCTGACACTGTCCTGCTGACATCCAAAGCATCTGATACAGCAGTTCCTGAGCAGAACAAAACAACAAAAACAGGACAGATGGATGGAATACCCATGTCAATGAAGAATGAAATGCCCATCTCCCAACTACTGATGATCATCGCCCCCTCCTTGGGATTTGTGCTCTTCGCATTGTTTGTGGCGTTTCTCCTGAGAGGGAAACTCATGGAAACCTATTGTTCGCAGAAACACACAAGGCTAGACTACATTGGAGATAGTAAAAATGTCCTCAATGACGTGCAGCATGGAAGGGAAGACGAAGACGGCCTTTTTACCCTCTAACAACGCAGTAGCATGTTAGATTGAGGATGGGGGCATGACACTCCAGTGTCAAAATAAGTCTTAGTAGATTTCCTTGTTTCATAAAAAAGACTCACTTATTCCATGGATGTCATTGATCCAGGCTTGCTTTAGTTTCATGAATGAAGGGTACTTTAGAGACCACAACTTCTCTGTCAAAAA

1. A method of treating cancer in a subject, the method comprisingadministering to the subject an effective amount of a PD-1 antagonistand a TIM-4 antagonist.
 2. The method of claim 1, wherein the TIM-4antagonist is selected from the group consisting of a ligand, antibody,and multivalent agent. 3-4. (canceled)
 5. The method of claim 1, whereinthe PD-1 antagonist is selected from the group consisting of a ligand,antibody and multivalent agent.
 6. The method of claim 5, wherein thePD-1 antagonist is an anti-PD-1 antibody comprising the CDR1, CDR2 andCDR3 domains in a heavy chain variable region having the sequence setforth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3 domains in a lightchain variable region having the sequence set forth in SEQ ID NO:
 15. 7.The method of claim 5, wherein the PD-1 antagonist is an anti-PD-1antibody comprising: (a) a heavy chain variable region CDR1 having thesequence set forth in SEQ ID NO: 17; (b) a heavy chain variable regionCDR2 having the sequence set forth in SEQ ID NO: 18; (c) a heavy chainvariable region CDR3 having the sequence set forth in SEQ ID NO: 19; (d)a light chain variable region CDR1 having the sequence set forth in SEQID NO: 20; (e) a light chain variable region CDR2 having the sequenceset forth in SEQ ID NO: 21; and (f) a light chain variable region CDR3having the sequence set forth in SEQ ID NO:
 22. 8. The method of claim5, wherein the PD-1 antagonist is an anti-PD-1 antibody comprising heavyand light chain variable regions having the sequences set forth in SEQID NOs: 13 and 15, respectively.
 9. The method of claim 5, wherein thePD-1 antagonist is an anti-PD-1 antibody comprising heavy and lightchains having the sequences as set forth in SEQ ID NOs: 11 and 12,respectively.
 10. The method of claim 5, wherein the PD-1 antagonist isan anti-PD-L1 antibody comprising the CDR1, CDR2 and CDR3 domains in aheavy chain variable region having the sequence set forth in SEQ ID NO:1, and the CDR1, CDR2 and CDR3 domains in a light chain variable regionhaving the sequence set forth in SEQ ID NO:
 3. 11. The method of claim5, wherein the PD-1 antagonist is an anti-PD-L1 antibody comprising: (a)a heavy chain variable region CDR1 having the sequence set forth in SEQID NO: 5 (b) a heavy chain variable region CDR2 having the sequence setforth in SEQ ID NO: 6; (c) a heavy chain variable region CDR3 having thesequence set forth in SEQ ID NO: 7; (d) a light chain variable regionCDR1 having the sequence set forth in SEQ ID NO: 8; (e) a light chainvariable region CDR2 having the sequence set forth in SEQ ID NO: 9; and(f) a light chain variable region CDR3 having the sequence set forth inSEQ ID NO:
 10. 12. The method of claim 5, wherein the PD-1 antagonist isan anti-PD-L1 antibody comprising heavy and light chain variable regionshaving the sequences set forth in SEQ ID NOs: 1 and 3, respectively.13-16. (canceled)
 17. The method of claim 1, wherein (a) the PD-1antagonist is an antibody comprising the CDR1, CDR2 and CDR3 domains ina heavy chain variable region having the sequence set forth in SEQ IDNO: 13, and the CDR1, CDR2 and CDR3 domains in a light chain variableregion having the sequence set forth in SEQ ID NO: 15; and (b) the TIM-4antagonist is an antibody.
 18. The method of claim 1, wherein (a) thePD-1 antagonist is an antibody comprising the CDR1, CDR2 and CDR3domains in a heavy chain variable region having the sequence set forthin SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a light chainvariable region having the sequence set forth in SEQ ID NO: 3; and (b)the TIM-4 antagonist is an antibody.
 19. The method of claim 1, whereinthe antagonists are formulated for intravenous administration.
 20. Themethod of claim 1, wherein the PD-1 antagonist is administered prior toadministration of the TIM-4 antagonist.
 21. The method of claim 1,wherein the TIM-4 antagonist is administered prior to administration ofthe PD-1 antagonist.
 22. The method of claim 1, wherein the TIM-4antagonist and the PD-1 antagonist are administered simultaneously. 23.The method of claim 1, wherein the cancer is a cancer selected from thegroup consisting of leukemia, lymphoma, blastoma, carcinoma and sarcoma,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, colorectal cancer, glioblastoma and colon carcinoma. 24.(canceled)
 25. The method of claim 1, which comprises administration ofan additional therapeutic agent. 26-27. (canceled)
 28. A compositioncomprising a PD-1 antagonist and a TIM-4 antagonist.
 29. (canceled) 30.A kit for treating a cancer in a subject, the kit comprising: (a) a doseof a PD-1 antagonist; (b) a dose of a TIM-4 antagonist; and (c)instructions for using the PD-1 antagonist and TIM-4 antagonist in themethod of any one of claim 1.